Lower respiratory tract infection (LRTI) is one of the leading cause of death in children under 5 years old around the world between 1980 and 2016. Distinguishing between viral and bacterial infection is challenging when children suffered from LRTI in the absence of pathogen detection. The aim of our study is to analyze the difference of serum β2-microglobulin (β2-MG) between viral LRTI and bacterial LRTI in children.This retrospective study included children with LRTI caused by a single pathogen from Yancheng Third People’s Hospital, Yancheng, China, between January 1, 2016 and December 31, 2019. Participants were divided into the younger group (1 year old ≤ age < 3 years old) and the older group (3 years old ≤ age < 5 years old) for subgroup analysis.Mycoplasma pneumoniae group. Compared with Streptococcus pneumoniae infection group, the serum β2-MG level of respiratory syncytial virus, influenza A virus and influenza B virus groups were significantly higher in children between 1 and 3 years old.A total of 475 children with LRTI caused by common respiratory pathogens were identified. In the younger group as well as the older group, the serum level of β2-MG in respiratory syncytial virus, influenza A virus and influenza B virus groups were significantly increased compared to that in the The serum β2-MG may distinguish viral infection from bacterial infection in children with LRTI. Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus)  is a common disease in children, and it also brings economic burden to families. The pathogenic microorganisms that caused LRTI mainly include viruses  and bacteria ( aureus) . LRTI is aureus) . LRTI in aureus) . It was eumoniae . Both upnfection . Howeverears old . Elevateears old . Howeverced LRTI .Rapid identification of pathogens is helpful for the treatment of LRTI. Early identification of viral infection can avoid overuse of antibiotics. The specificity of culture is high, but it takes a long time. The sensitivity of serological diagnosis depends on the time of sample collection . The polβ2-MG is a nonglycosylated protein (11.6 kDa), which is found on the surface of almost all nucleated cells . β2-MG iM. pneumonia and Streptococcus pneumonia. In this study, the distribution difference of β2-MG in viral infection and bacterial infection was analyzed. We discussed the theoretical basis of β2-MG in distinguishing viral infection from bacterial infection. It was expected that the detection of β2-MG may provide help for the management of respiratory tract infection diseases, and antibiotic management.Is there a better biomarker to identify viral infection in LRTI? We retrospectively studied the serum levels of β2-MG in patients with LRTI caused by respiratory syncytial virus, influenza A virus, influenza B virus M. pneumonia and Streptococcus pneumonia. Blood samples, sputum samples and nasopharynx swabs were collected and sent to the laboratory for pathogen detection within 24 h after admission.We reviewed the electronic medical records of all hospitalized children with a single pathogen of LRTI from Yancheng Third People’s Hospital, Yancheng, China, between January 1, 2016 and December 31, 2019. The LRTI in this study consisted of acute bronchitis and community-acquired pneumonia. The diagnosis of LRTI is based on the following criteria: diagnosis of acute bronchitis according to persistent cough less than 3 weeks and lack of radiologically visible infiltrates in lungs or other underlying lung disease. Diagnosis of community-acquired pneumonia according to fever, lung radiologically visible infiltrates, cough, sputum production, shortness of breath, abnormal breath sounds and rales in auscultation of lung. Pathogens include respiratory syncytial virus, influenza A virus, influenza B virus, M. pneumoniae, respiratory syncytial virus, influenza A virus, influenza B virus, Streptococcus pneumoniae. (4) β2-MG is often used as an index to evaluate renal function (Inclusion criteria: (1) Children suffered from LRTI. (2) There was only one pathogen causing LRTI. (3) Pathogens included function . The renfunction . TherefoExclusion criteria: (1) Children had primary immunodeficiency disease or kidney disease. (2) Immunodepressant was used in the last 2 weeks.This study was approved by the ethics committee of the Yancheng Third People’s Hospital . Individual informed consent was waived by the ethics committee of the Yancheng Third People’s Hospital because the study was considered to pose the least risk to participants.M. pneumoniae infection: During hospitalization, the serum immunoglobulin M antibody of M. pneumoniae changed from negative to positive. Streptococcus pneumoniae infection: Sputum cultures or blood cultures were positive.Viral infection: The diagnosis of respiratory syncytial virus, influenza A virus and influenza B virus infection were based on antigen detection or PCR nucleic acid detection of nasopharynx swab samples. t-test. χ2-test was used to compare the difference of count data in different groups. Due to the different age distribution of different diseases, subgroup analysis of β2-MG was carried out according to age. All analysis were performed through SPSS 24 software.Categorical variables were described by frequencies and percentage. Continuous variables features were expressed by mean ± standard deviation or interquartile ranges. The difference between the two groups of continuous variables is analyzed by student’s M. pneumoniae infection and 14 cases of Streptococcus pneumonia infection. The epidemiological and clinical characteristics of patients were shown in Streptococcus pneumonia. In the older group (3 years old ≤ age < 5 years old), it is mainly 80.4% of children affected by M. pneumoniae and 62.7% of children affected by influenza B virus.We collected 475 children with LRTI caused by single pathogen . There wM. pneumoniae infection. There were only three cases of lower respiratory tract infection with Streptococcus pneumoniae in children between 3 and 5 years old, so there was no statistical comparison. The concentration of serum β2-microglobulin was 1.67 mg/L, 1.69 mg/L and 1.51 mg/L, respectively. The β2-MG concentration in viral infection group was compared with that in Streptococcus pneumoniae infection group. The β2-MG concentration was significantly increased in respiratory syncytial virus group, influenza A virus group and influenza B virus group (us group . The serus group .M. pneumoniae. The results suggest that serum β2-MG may be helpful to distinguish M. pneumoniae infection from viral infection in LRTI. M. pneumoniae pneumonia. β2-MG freely passes through the glomerular filtration barrier. Urinary β2-MG increased when serum β2-MG level exceeded renal threshold. It was found that macrophages infected with influenza A virus could temporarily increase the expression of β2-MG (In our research, children with LRTI caused by respiratory syncytial virus, influenza A virus and influenza B virus had higher levels of serum β2-MG than children with LRTI caused by of β2-MG . Our resStreptococcus pneumoniae group between 1 and 3 years old. The serum β2-MG level of respiratory syncytial virus group, influenza A virus group and influenza B virus group were higher than that of the Streptococcus pneumoniae group. Previous studies had found that the increased concentration of serum β2-MG was related to human cytomegalovirus, Epstein-Barr virus, hepatitis C virus and hepatitis B virus (We analyzed virus groups and  B virus . The lev B virus . OverallCorrelation analysis showed that the serum β2-MG level decreased with the increase of gestational age in normal fetuses . We analM. pneumoniae, therefore the generalisability of these findings to other bacterial infections is limited. Fourth, we did not perform any multivariate linear regression models to see if viral/non-viral atiology was an independent predictor of higher β2-MG levels, after including possible confounders such as age, creatinine/urea nitrogen levels, gender, C-reactive protein, white blood cell. Fifth, we lack analysis of healthy children.There are some obvious limitations in our study, and the results may be biased. First, this retrospective study collected a small number of samples. Second, although we had screened cases with a single pathogen infection, the limitations of clinical pathogen detection may be combined with other undetected pathogens. Third, the vast majority of the bacterial cases in this study were of M. pneumoniae group and Streptococcus pneumoniae group. Hence we infer that serum β2-MG may play an important role in the prediction of viral LRTI in children. It may provide a basis for avoiding antibiotic abuse. The molecular biological mechanism of β2-MG needs further verification and exploration.In a retrospective analysis of the children with lower respiratory tract infection, we found that serum β2-MG levels in respiratory syncytial virus, influenza A virus, and influenza B virus groups were higher than 10.7717/peerj.9814/supp-1Supplemental Information 1Click here for additional data file.


Stigma is the deleterious, structural force that devalues members of groups that hold undesirable characteristics. Since stigma is created and reinforced by society—through in-person and online social interactions—referencing the novel coronavirus as the “Chinese virus” or “China virus” has the potential to create and perpetuate stigma.The aim of this study was to assess if there was an increase in the prevalence and frequency of the phrases “Chinese virus” and “China virus” on Twitter after the March 16, 2020, US presidential reference of this term.Using the Sysomos software , we extracted tweets from the United States using a list of keywords that were derivatives of “Chinese virus.” We compared tweets at the national and state levels posted between March 9 and March 15 (preperiod) with those posted between March 19 and March 25 (postperiod). We used Stata 16 (StataCorp) for quantitative analysis, and Python (Python Software Foundation) to plot a state-level heat map.A total of 16,535 “Chinese virus” or “China virus” tweets were identified in the preperiod, and 177,327 tweets were identified in the postperiod, illustrating a nearly ten-fold increase at the national level. All 50 states witnessed an increase in the number of tweets exclusively mentioning “Chinese virus” or “China virus” instead of coronavirus disease (COVID-19) or coronavirus. On average, 0.38 tweets referencing “Chinese virus” or “China virus” were posted per 10,000 people at the state level in the preperiod, and 4.08 of these stigmatizing tweets were posted in the postperiod, also indicating a ten-fold increase. The 5 states with the highest number of postperiod “Chinese virus” tweets were Pennsylvania (n=5249), New York , Florida , Texas , and California . Adjusting for population size, the 5 states with the highest prevalence of postperiod “Chinese virus” tweets were Arizona (5.85), New York (6.04), Florida (6.09), Nevada (7.72), and Wyoming (8.76). The 5 states with the largest increase in pre- to postperiod “Chinese virus” tweets were Kansas , South Dakota , Mississippi , New Hampshire , and Idaho .The rise in tweets referencing “Chinese virus” or “China virus,” along with the content of these tweets, indicate that knowledge translation may be occurring online and COVID-19 stigma is likely being perpetuated on Twitter. Stigma is the deleterious, structural force that devalues those who hold undesirable characteristics . Stigma On March 16, 2020, the president of the United States referred to the novel coronavirus as the “Chinese virus” on Twitter. He tweeted “The United States will be powerfully supporting those industries... that are particularly affected by the Chinese Virus...” After this presidential reference, a dialogue emerged examining if the phrase “Chinese virus” was xenophobic and stigmatizing, considering the availability of alternative scientific names such as coronavirus or COVID-19. Since stigma is created and perpetuated by society through social interaction and public commentary , and stigma is reinforced by those in power , we hypothesized that there would be an increase in the frequency of the phrases “Chinese virus” and “China virus” on Twitter, comparing the prevalence of these phrases before and after the presidential reference.Twitter is an online social media platform where users send and receive short posts (maximum 280 characters) called tweets. Twitter currently has 152 million daily users, who produce about 500 million daily tweets .We downloaded tweets from all 50 US states, using the Sysomos software . We extracted tweets that mentioned “Chinese virus” or “China virus” but did not contain “COVID-19” or “coronavirus.” The list of keywords referencing the “Chinese virus” are “Chinesevirus,” “Chinese virus,” “Chinavirus,” “China virus,” “#ChineseVirus19,” “#Chinesevirus,” “#ChineseVirusCorona,” and “#Chinavirus.” We excluded tweets containing the keywords “coronavirus,” “corona virus,” “COVID-19,” “COVID19,” “#COVID2019,” and “#corona.” By excluding tweets that contained both “Chinese virus” and “coronavirus,” we collated a sample of tweets that represented the intent of using “Chinese virus” in place of a scientific alternative, likely indicating deliberate stigmatization. We imputed the location of tweets based on Twitter users’ self-reported state of residence. Tweets posted between March 9 and March 15, 2020 (preperiod), were compared with tweets posted between March 19 and March 25, 2020 (postperiod). Original tweets and quote tweets (adding comments to an existing tweet) were included but not retweets (reposting of an existing tweet). Our final sample  contained all tweets posted in the pre- and postperiods by US-based Twitter users that exclusively mentioned a derivative of “Chinese virus.” Data extraction was conducted on April 10, 2020. Ethical approval was provided by the University of Alabama at Birmingham Institutional Review Board (IRB-#300005071).We used Stata 16 (StataCorp) to analyze our Twitter data and Python software (Python Software Foundation) to plot our state-level gradient heat map.A total of 16,535 “Chinese virus” or “China virus” tweets were identified in the preperiod, and 177,327 tweets were identified in the postperiod, illustrating a 972.43%  increase. Comparatively, the number of tweets referencing COVID-19 in the preperiod and postperiod remained steady, at about 4.9 million tweets per period. A total of 13,569 (82.06%) of the preperiod and 145,521 (82.06%) of the postperiod tweets were associated with a Twitter user’s self-reported US state. . State population sizes were taken from the 2019 US Census Bureau estimates  narrative. It's the #WuFlu #ChineseCoronaVirus #ChinaVirus”“#ChinaVirus #ChinaLiesPeopleDie”The pandemic is currently underway, so Twitter data—both in quantity (quantitative) and content —are rapidly shifting. We were unable to screen for automatically generated tweets (bots) within this short report ,14. GeogThere is a growing body of academic literature that leverages Twitter data to assess trends in population health and public sentiment -17. ChewFuture research could evaluate and show that stigma mechanisms work online, validate if Twitter and social media data can be informative to epidemic surveillance and health communication, examine the extent that Twitter and social media data is reliable in informing public health efforts and social science research, and explore how Twitter users view COVID-19 and the COVID-19 public health response .Additionally, although there is a growing body of research using tweets to examine aspects of the novel coronavirus -22, to oThe rise in tweets citing “Chinese virus” or “China virus” instead of COVID-19 or the novel coronavirus after the presidential reference on Twitter, along with the content of these tweets, indicate that knowledge translation may be occurring online and COVID-19 stigma is likely being perpetuated on Twitter. Generally speaking, perpetuating COVID-19-related stigma by using the phrase “Chinese virus” could harm public health efforts related to addressing the pandemic, specifically inciting fear and increasing distrust of public health systems by Chinese and Asian Americans. If these stigmatizing terms persist as malicious synonyms for the novel coronavirus, reparative efforts may be required to restore trust by marginalized communities.


Viral infections and associated diseases are responsible for a substantial number of mortality and public health problems around the world. Each year, infectious diseases kill 3.5 million people worldwide. The current pandemic caused by COVID-19 has become the greatest health hazard to people in their lifetime. There are many antiviral drugs and vaccines available against viruses, but they have many disadvantages, too. There are numerous side effects for conventional drugs, and active mutation also creates drug resistance against various viruses. This has led scientists to search herbs as a source for the discovery of more efficient new antivirals. According to the World Health Organization (WHO), 65% of the world population is in the practice of using plants and herbs as part of treatment modality. Additionally, plants have an advantage in drug discovery based on their long-term use by humans, and a reduced toxicity and abundance of bioactive compounds can be expected as a result. In this review, we have highlighted the important viruses, their drug targets, and their replication cycle. We provide in-depth and insightful information about the most favorable plant extracts and their derived phytochemicals against viral targets. Our major conclusion is that plant extracts and their isolated pure compounds are essential sources for the current viral infections and useful for future challenges. A virus is a tiny parasite that has no capacity to replicate itself. Once infected in a host agent or living cell, it produces more viruses using host machinery. With their complexity and diversity, it survives for a long time in the host, bypassing the treatments, and it causes devastating issues such as pandemics . They haViral infections and associated diseases are responsible for a substantial number of mortality and public health problems around the world. Each year, infectious diseases kill 3.5 million people worldwide ,7. The dHence, the aim of the present retrospective review is an update on the discovery regarding different plants and lead compounds isolated from them against the essential and clinically significant virus such as the human immunodeficiency virus (HIV), herpes simplex virus (HSV), influenza, and hepatitis c, clarifying their indication with viruses and mechanisms of action. http://www.theplantlist.org and http://www.ipni.org websites. To obtain the appropriate literature, we have used relevant keywords such as plants, viruses, phytochemicals, HIV, HSV, influenza, hepatitis-c, HIV integrase, HIV-reverse transcription, HIV-protease, virucidal action, virus replication imbibition, viral attachment, inhibition of hemagglutination, HCV infection replication, etc. These keywords were searched in relevant databases such as Google Scholar, PubMed, Scopus, Scielo, etc. We have collected information from research articles, review articles, PhD theses, books, chapters, and conference abstracts from 1975 to 2020. A total of 207 species have been reported in this review article. The taxonomy of the plant species was properly identified from The main target of the human immunodeficiency virus (HIV) is our immune system, where it affects and destroys the immune system function. At present, more than 35 million people are suffering from HIV; so far, it caused more than 39 million HIV-related deaths worldwide . The ove+ T cells [+ lymphoid cell surface. After the viral capsid enters the cell, reverse transcriptase liberates a positive sense single-stranded RNA, coping it into a complementary DNA. Then, the nuclei of host cells become integrated with the viral RNA. The integrated DNA is then transcribed into RNA in the presence of transcription factors such as NF-kB, which is then spliced into messenger RNA (mRNA) [HIV is a member of the genus Lentivirus, part of the family Retroviridae . HIV con T cells ,15. ThisA (mRNA) ,17,18. TThe antiviral treatments explicitly target these key areas of virus multiplication. Nonetheless, the infection rate of HIV is increasing in spite of ART . MoreoveNatural products have been explored in finding anti-HIV agents with a critical focus in four mechanisms. They are HIV integrase strand transfer inhibitors , NucleosIn our search for natural products in the mentioned databases, we have observed that the majority of the natural products are evaluated for anti-HIV properties up to the crude extraction level only. So, we found a few major secondary metabolites isolated from plants, which have good activity against HIV. A list of plant species with inhibition studies is summarized in The screening of medicinal plants has delivered plenty of secondary metabolites with anti-HIV properties. They include alkaloids, triterpenoids, flavonoids, coumarins, phenolics, tannins, saponins, phospholipids, xanthones, quinones, etc. . There iThe herpes simplex virus (HSV) infection, otherwise known as genital herpes (GH), is the most frequent cause of genital ulceration worldwide. In general, herpes can appear commonly in the mouth and genitals. The primary cause of oral herpes is the HSV-1 type strain, but genital herpes is commonly caused by the HSV-2 type strain . HSV-serHSV is a member of Herpesviridae, which is a large family of enveloped double-stranded DNA viruses that causes diseases in both human and animals . Even thThere is no ultimate cure for HSV, but the current strategies are mainly focused on symptomatic relief. Both innate and adaptive immune systems can control HSV infections. In fact, the nature of HSV infection is dependent upon how the virus bypasses the host innate immune system. In the current system of practice, antiviral drugs are classified as virucidal, immunomodulators, and chemotherapeutic agents . There iThe natural products always served as a trustable source for new compounds with antiviral properties. Many studies have been carried out since 1995 to isolate bioactive antiviral compounds from plants and functional foods. Accordingly, a large number of plant-derived anti-HSV drugs have been described in several studies. A list of plant species with inhibition studies is summarized in 50 dose.Many herbal compounds have been investigated in the past for their effectiveness against HSV. The purification of new lead compounds from the plants and evaluating their targets and mechanism of action in HSV is also equally important. Many secondary metabolites have been proven to have anti-HSV effects such as lignans, tannins, saponins, terpenes, alkaloids, quinones, and glucosides ,157,158.Pandemics are the mainly remarkable appearances of the influenza virus . Three pInfluenza virus belongs to Orthomyxoviridae family (RNA viruses), which includes seven genera  ,170. AlpInfluenza virus mainly targets the columnar epithelial cells in the respiratory tract . FirstlyIn the contingency of a flu pandemic with a new strain, antiviral drugs symbolize the primary line of defense ,182. Res50 dose.Among viral infections, the viruses of the influenza viral infection have the ability to mutate their genome and become resistant to drugs . Thus, tHepatitis C virus (HCV) infection is considered as a significant public health problem. It has infected around 180 million people worldwide . In deveHepatitis C virus belongs to the Hepacivirus genus of the Flaviviridae family. It is a small enveloped virus with single-stranded genomic RNA with two embedded viral glycoproteins . In the There are synthetic agents available now against HCV, but they have a lack of specific treatment for HCV therapy. Another concern in these cases is the presence of severe side effects and reported poor response rates. To manage and to get these problems under control for better treatment against HCV, new potential agents to be explored. As we see in the cases of other viruses discussed in the review, there are many promising natural products, which have led to the discovery of potent HCV inhibitors. A list of plant species with inhibition studies is summarized in Developing an anti-HCV drug has become an important priority due to the complexity of the disease. Natural compounds always serve as a lead to create new drugs. There is a substantial increase in the reports on phytochemicals that show anti-HCV properties. Both primary and secondary metabolites have shown promising activities. For instance, alkaloids, flavonoids, polyphenols, coumarins, and peptides have been reported to possess anti-HCV activities . We haveViral infections and pandemic have been recorded as a potential risk for human survival. The lack of proper prophylactic vaccines and drugs for many viruses makes the situation worse in health management. There is a great need for novel antiviral compounds for drug development. This review provides in-depth and insightful information about different species of plants and their families with significant secondary metabolites with evidence-based antiviral properties. Based on the literature, we provided very promising drug candidates that have been investigated through in vitro screening, and cellular targets have been observed. In the current review, we have selected HIV, HSV, HCV and Influenza virus. Looking at the spectrum of plants and isolated compounds, we have seen that there is no significant selectivity among the plants and their compounds in inhibiting DNA or RNA virus. We have found that a similar class of phytochemicals can inhibit both types, but with the ability to inhibit different sites of mechanism. However, these compounds need a lot of further investigation to make them appropriate for clinical use. The pace of new antiviral drugs from natural origin has experienced a substantial upsurge in the last decade. Natural products directly or indirectly support the drug discovery against viruses. Many anti-viral drugs has been discovered from a synthetic source, but originally modeled on a natural product parent structure. Most of the plants we have identified in this review hold other pharmacological benefits, proven long ago, together with their safety profile. This promotes the acceptance of these plants and their phytochemicals for antiviral drug discovery and development programs. Nevertheless, a thorough purification process for identifying new lead compounds and their preclinical and safety testing is a prerequisite. The current COVID-19 pandemic has taught us a more significant lesson: it is difficult to survive in this earth without accepting the probability of more pandemics in the future. Hence, taking the facts in a very comprehensive manner, a cohesive and focused drug discovery approach is warranted.


Notably, the level of virome diversity revealed in penguins is comparable to that seen in Australian waterbirds, including many of the same viral families. These data run counter to the idea that penguins are subject to lower pathogen pressure. The repeated detection of specific viruses in Antarctic penguins also suggests that rather than being simply spill-over hosts, these animals may act as key virus reservoirs.Despite its isolation and extreme climate, Antarctica is home to diverse fauna and associated microorganisms. It has been proposed that the most iconic Antarctic animal, the penguin, experiences low pathogen pressure, accounting for their disease susceptibility in foreign environments. There is, however, a limited understanding of virome diversity in Antarctic species, the extent of in situ virus evolution, or how it relates to that in other geographic regions. To assess whether penguins have limited microbial diversity we determined the RNA viromes of three species of penguins and their ticks sampled on the Antarctic peninsula. Using total RNA sequencing we identified 107 viral species, comprising likely penguin associated viruses ( Geographical separation and extreme climates have resulted in the long isolation of Antarctica and the subantarctic islands. The result is a unique assemblage of animals, some relying entirely on the frozen continent, with others utilising the fringes. Such geographic isolation has been proposed to explain why Antarctic fauna supposedly harbour a paucity of viruses, and is supported by the observation that captive Antarctic penguins are highly susceptible to infectious disease . It has Pygoscelis adeliae), Chinstrap penguins (Pygoscelis antarctica), and Gentoo penguins (Pygoscelis papua)  diversity were calculated for each library at the virus family and genus levels using the Rhea script sets , and comvegan  and phylpackages .We characterised the transcriptomes of six libraries comprising ten individuals each, corresponding to three Antarctic penguin species in three locations and four tick libraries comprising a total of 20 ticks Table . RNA seqThe abundance of RSP13, a stably expressed host reference gene in the avian colon , was simThe abundance of avian viral reads was the highest in Chinstrap penguins sampled on Kopaitik Island , and the lowest in Gentoo penguins sampled at the GGV Base for which no avian viral reads were detected. Because this colony is comprised solely of Gentoo penguins only this species was sampled . Alpha dPicornaviridae were the most abundant in the Chinstrap and Adelie penguin libraries, comprising 99, 32, 83, and 71% of all the avian viral reads in these four libraries. In marked contrast, the Picornaviridae comprised only 0.25% of avian viral reads from Gentoo penguins on Kopaitik Island (and no avian viruses were found in the Gentoo penguins from GGV). Beta diversity demonstrated connectivity in the RNA viromes from the Adelie penguins, driven by a number of shared viral species across the Kopaitik Island and King George Island sampling locations that are 130 km apart , were identified in the penguin and tick libraries Fig. . Of thesae Figs.  and 3 . As these 82 viruses are unlikely to be associated with penguins or their ticks, they are not described further.Strikingly, we identified 82 divergent novel virus species in the penguin libraries that clustered phylogenetically within 11 defined families, as well as three viruses that clustered with a group of unclassified viruses. These unclassified viruses are likely associated with penguin diet or their microbiome: fish, invertebrates, plants, fungi and bacteria Fig. , Fig. 3.Nacovirus —a group dominated by avian viruses  and Adelie (Mawson virus) penguins. Shackleton virus falls as an outgroup to a clade of rotaviruses recently described in wild birds, which are themselves divergent from rotavirus G virus, while Mawson virus is a sister group to rotavirus D Fig. . Hilary Finally, although most of the novel viruses documented here had RNA genomes, we also identified a novel alphaherpesvirus, Oates virus, that falls as a sister group to Gallid and Psittacid hepervirus 1. Notably, this virus was distantly related to an alphaherpesvirus previously described in penguins  Fig. .Previous studies of Antarctic penguins have detected avian IAV and avian avulaviruses , 16, 21.We also identified a deltacoronavirus and an avastrovirus Figs. –S12. TheI. uriae ticks sampled here was a variant of Taggert virus, a nairovirus  previously identified in penguin associated ticks on Macquarie Island: the contigs identified in our data showed 81.6% nucleotide sequence similarity in the RdRp region to Taggert virus [Bunyavirales were also discovered—Ronne virus and Barre virus—both members of the Phenuiviridae I. uriae dae Fig.  that exhdae Fig. .Fig. 7PhIflaviridae-like, Alphatetraviridae, Reoviridae, Rhabdoviridae, and Levivirdae. A novel Ifla-like virus, Gerbovich virus, was identified within both nymph libraries. This virus clustered with a group of tick associated ifla-like viruses, including Ixodes holocyclus iflavirus and Ixodes scapularis iflavirus , Fennes virus, was identified in the adult male, female and nymph libraries, although we were only able to assemble four segments. Notably, Fennes virus falls basal to the existing coltivirus group, exhibiting just 30% amino acid sequence similarity to Shelly headland virus, recently identified in I. holocyclus ticks from Australia , and in wildlife. We aimed to assess whether Antarctic penguin colonies experience low pathogen pressure as a result of their geographic and climatic isolation, employing meta-transcriptomic virus discovery from three penguin species and their ticks. Although our sample size was relative small, we were able to demonstrate the presence of 13 viral species in three species of penguins from the Antarctic peninsula and nine in ticks associated with penguin nesting sites. These preliminary data therefore counter the idea that animals in the Antarctic harbour less microbial diversity than animals from other geographic regions. Indeed, the penguins sampled show similar levels of RNA virome diversity as Australian wild birds , 48. RecPicornaviridae, at very high abundance . Adelie penguins also had high viral diversity, with apparently healthy birds carrying three or four viral species. Perhaps more striking was that Adelie penguins on King George Island and the Antarctic peninsula shared viruses, despite the greater than 100 km distance between these colonies. A similar trend was observed by Wille et al. [Macronectes gigantes), Kelp Gulls (Larus dominicanus) or Skuas (Stercorarius spp.) [Notably, as all the penguins sampled appeared healthy, the disease-causing capacity of these viruses is uncertain. Ten Chinstrap penguins sampled on King George Island harboured five different viral species, mostly from the e et al.  who founus spp.) , 52, or Combined, these data strongly suggest that penguins are not merely spill-over hosts, but may be central reservoir hosts for a diverse range of viruses. This is apparent in two observations. The first is the repeated detection of specific virus species, such as avian avulaviruses, and that these viruses comprise distinct clusters of related variants. Antarctic penguins have been sampled since the 1970s, and avian avulaviruses have repeatedly been detected, both by serology and PCR. The detection of phylogenetically related avian avulavirus 17 in 2013 in Adelie penguins , in 2014Nairoviridae, while Fennes virus was a member of the Reoviridae. Taggert virus was originally identified in I. uriae collected from penguin colonies, and is one of seven I. uriae associated virus species identified in ticks collected from penguin colonies on Macquarie Island [Orthonairovirus, and close to the pathogenic arbovirus, Crimean-Congo Haemorrhagic fever virus. Interestingly, we not only identified Taggert virus in all four tick libraries, but also in Chinstrap penguins on Kopaitik Island. This strongly supports the idea that penguins acted as a reservoir host for Taggert virus [The second key observation that indicates that penguins are potential virus reservoirs was the presence of likely arboviruses, which is why we paired our analysis of the penguin RNA virome with that of the ticks that parasitise them. Of the nine species of viruses identified within the ticks in this study, two clustered phylogenetically with other arboviruses: the previously detected Taggert virus fell within the e Island . Taggertrt virus . In thisColtivirus that includes the pathogenic tick-borne virus Colorado tick fever virus as well as a number of tick associated viruses and a species identified in African bats [I. uriae from Macquarie Island in a series of three studies between the 1970s and 2009 belonged to just four families—Reoviridae, Narioviridae, Phenuiviridae, and Flaviviridae [Leviviridae.Also of note was Fennes virus that clustered phylogenetically within the genus can bats , 53–55. iviridae –25—threeNarnaviridae and Leviviridae. Given their phylogenetic position these viruses are likely associated with the fish, crustacean, and plant species ingested by the penguins as a part of their diet, as well as infecting unicellular parasites, fungi, and bacteria. A number of these viruses may also be associated with penguin gut flora: indeed, cloacal swabs are used extensively in studies of bird gut microbiomes [Narnaviridae are known to infect fungi and protists, while the related Leviviridae infect bacteria [Nodaviridae and Tombusviridae, associated with both vertebrate and invertebrate infecting viruses in the Picornavirales [Picobirnaviridae/Partitiviridae group. While the Partitiviridae are recognised as invertebrate associated viruses, the host association of the Picobirnaviridae is currently uncertain [There was extensive diversity of viruses identified in the penguin samples that were likely to be associated with hosts other than birds, including entire clades of novel viruses within phylogenetic trees of the robiomes , 57. Duebacteria –61. Otheavirales . There wncertain . OverallIn sum, we reveal substantial viral diversity in Antarctic penguins, their diet and their ticks. We therefore expect that additional viruses will be identified with increased sampling, and a wider variety of sample types, reflecting what it is in reality a relatively high diversity of unique fauna and flora on the Antarctic continent. Clearly, additional sampling of penguins and other species in Antarctica is critical to elucidate the epidemiological connection between Antarctica and the rest of the globe, and from this better understand the mechanisms of viral introduction and circulation.Supplemental MaterialSupplemental Material Table S2


Nearly all lethal viral outbreaks in the past two decades were caused by newly emerging viruses. Virusesare often studied by electron microscopy (EM), which provides new high-resolution data on the structureof viral particles relevant to both fundamental virology and practical pharmaceutical nanobiotechnology. Electronmicroscopy is also applied to ecological studies to detect viruses in the environment, to analysis of technologicalprocesses in the production of vaccines and other biotechnological components, and to diagnostics.Despite the advances in more sensitive methods, electron microscopy is still in active use for diagnostics. Themain advantage of EM is the lack of specificity to any group of viruses, which allows working with unknownmaterials. However, the main limitation of the method is the relatively high detection limit (107 particles/mL),requiring viral material to be concentrated. There is no most effective universal method to concentrate viruses.Various combinations of methods and approaches are used depending on the virus and the goal. A modernvirus concentration protocol involves precipitation, centrifugation, filtration, and chromatography. Here wedescribe the main concentrating techniques exemplified for different viruses. Effective elution techniques arerequired to disrupt the bonds between filter media and viruses in order to increase recovery. The paper reviewsstudies on unique traps, magnetic beads, and composite polyaniline and carbon nanotubes, including thoseof changeable size to concentrate viral particles. It also describes centrifugal concentrators to concentrateviruses on a polyethersulfone membrane. Our review suggests that the method to concentrate viruses andother nanoparticles should be chosen with regard to objectives of the study and the equipment status of thelaboratory. Electron microscopy (EM) is often used to study viruses.This method makes it possible to determine the shapes andsizes of viral particles and their localization within cells, aswell as to identify cytological changes in cells. The mainadvantage of EM is the lack of specificity to any groupof viruses, as opposed to immunological and moleculartests. With the present advances in genetic engineeringand the use of a wide range of pseudovirus-based assays invaccine development, the list of possible research targetsfor EM and the scope of problems solved have expandedsignificantly. A valid EM study requires that the sampleshould contain enough viral particles. It was shown earlier that EM visualization requires virioncontents no lower than 107 particles/mL. Thus, the necessityfor purification and concentration of viral materialarises rather often. It is noteworthy that EM is also necessaryfor the refinement of virus concentration methods.Moreover, in the age of nanotechnology we cannot ignorethe fact that transmission EM (TEM) is used to control thefabrication of nanoparticles and their application .In addition to the classical application of the method, i. e.studying structures of viruses, infiltration processes, andvirus transmission in organisms, EM is also used in at leastthree fields of applied research: diagnostic EM (DEM);ecological EM (detection of viruses in the environment),and problems associated with technological processes(manufacturing of vaccines and other components) thatuse biotechnological methods to yield the products andrequire that the product should be proven to be virus-free.Despite the advances in more sensitive methods, such aspolymerasechain reaction (PCR) or immunoenzyme analysis(IEA), EM still remains a widely applicable diagnostictool .The main advantage of EM in diagnostics of infectious diseasesis its versatility, i. e. the ability to observe ‘anything’in a single sample without prior knowledge of possiblemicroorganism identities present in the sample . Another advantage of EM is its fastness,as it takes as little as 10–15 minutes for a wide range ofobject sizes to be studied. EM makes it possible to analyzeboth liquid and tissue samples and requires no additionalinformation, unlike the diagnostic methods using nucleicacids (PCR) or antibodies (IEA).The main limitation of DEM is the virion content insamples. To overcome this limitation, various concentration techniques are used, such as ultracentrifugation, ultrafiltration,examination of ultrathin sections, etc. In , the authors describe virus concentration byfiltration straight onto EM support grid with holey carbonsubstrate Quantifoil R1/4 . This technique combined withscanning EM (SEM) improves the virus detection thresholdto contents of 102 viral particles per 1 mL.Thus, DEM is a valuable method for observing newlyemerging diseases and potential bioterrorism agents. Finally,ultrastructural studies performed using EM providedetailed examination of virus morphogenesis, virus – cellinteractions, and pathogenetic aspects of viral infectionsincluding the development of preventives and treatments.The use of EM for ecological purposes (detection of virusesin the environment) appears to be an obvious solution, butit is rarely used in practice. When it is, its most commontarget is the pollution of water resources, which suppliesalmost illimited volumes of research material. One of thefew papers dealing with seawater as a research subject waspublished in 1999 . The described concentrationprocess included two stages: concentration of avast amount of seawater using a tangential flow filtrationsystem and ultrafiltration using a centrifugal concentratorwith further visualization by means of TEM. The samemethods were used later in . The authorsstudied over 150 L of seawater from the Yangshan Deep-Water Port area . However,these days EM is quite rarely used for such analytical tasks,as they are mostly solved by more sensitive methods, firstof all, PCR. The reason is the limited range of possibletargets, which are being typically presupposed or knownbeforehand, so PCR is often substituted for EM at the stagewhen an infectious agent has to be detected.7 particles/ mL),ultracentrifuge concentration in a discontinuous sucrosedensity gradient with EM (105 particles/mL), and themethod involving a thin section of a homogenous matrixof viral particles, cell debris, agar medium (sensitivity105 particles/mL). Routine EM is obviously not suitablefor registering virus contamination.Vaccines are among the most effective pharmaceutical productsin public healthcare showing great results in terms ofsafety. Since biological materials are involved in vaccineproduction, the process must be protected from sporadicalcontamination. The necessity for controlling biotechnologicallyproduced materials is broadly discussed . The production of virus vaccines and other cellularbiopharmaceutical products is a complex technologicalprocess, which involves various biological materials . The whole process is potentially vulnerable to contaminationby foreign agents that may be unintentionally brought inwith the materials used or from environmental sources. Anexample of this type of study may be found in , where three methods for retrovirus contaminationassessment in mouse culture supernatants and СНО celllines were compared: direct count of viral particles in a solutionwith latex beads  from tobacco mosaic virus was controlled by TEM.It was shown that SP could be visualized via TEM withoutcontrasting agents .There is no most effective universal method to concentrateviruses. Various combinations of methods and approachesare used depending on the virus and the problem set. Virusconcentration processes currently include sedimentation,centrifugation, filtration, and chromatography .Virus precipitation is commonly achieved with polyethyleneglycol (PEG), ammonium sulfate, or calcium phosphate.It is a convenient tool for obtaining viruses in bothlarge and small quantities, while also providing the advantageof virus separation from most heterologous proteins.For example, the precipitation and purification methodusing PEG 6000 with further ultracentrifugation and clarificationwidely used for influenza viruses was successfullyapplied for concentration and purification of avian influenzaType A virus (ten different strains) from virus-containingsuspensions . Another paper presented data on purificationand concentration of horse rhinopneumonia (HRP) virususing various techniques. It was found that concentrationof HRP virus using PEG-6000 with 0.5 М sodium chloridewith subsequent dialysis yields concentrates with high infectiousand antigen activity. It was shown that adsorptionin presence of PEG produced virus preparations suitablefor manufacturing inactivated HRP vaccine.Polyethylene polyamine (PEPA) with a mass of 16,000 Dwas shown to be more suitable for concentration of FMDvirus and porcine enterovirus (serotype 7) by precipitationthan PEG-6000 . It was found that the most complete precipitation of FMD virus was achievedat PEPA contents of 0.1 and 0.2 %. There, virus losseswere 0.74 and 0.11 %, respectively. It was also shown thatvaccines produced using the preparations obtained via twodifferent precipitation techniques were different in terms oftheir efficacy. The PEPA-based technique was preferablesince the respective vaccine was three times as effective.Centrifugation is an easy-to-use large-scale separation methodbased on differences in density. Due to small sizes ofviral particles, high speed and centrifugal force are required,which, however, can make the particles noninfectious. Centrifugation in density gradientusing sucrose, cesium chloride (CsCl), or iodixanol is aviable alternative. The use of sucrose provides a viscoushyperosmotic solution. Density gradient of iodixanol is alow-viscosity system capable of producing isotonic solutionand maintaining virus functionality . Itwas shown  that the use of iodixanolgradient provides 37 % retrovirus recovery with a promisingpurity of 95 %. To separate horse influenza virusfrom virus-containing allantoic fluid, ultracentrifugationin sucrose density gradient and DEAE cellulose-basedion exchange chromatography were used . It was found that complete virus adsorption byion exchanger occurred in a buffer solution with рН 7.0,whereas elution required a buffer with 0.5 М sodium chloridesolution рН 7.4. As a result, virus preparations wereobtained with ballast protein removal of 98 %.7and 3.4 × 108 copies/mL.To concentrate enteroviruses from various water bodies, aunified membrane filtration method was developed usingthe microfiltration (MF) mode of the MFM 0142 moduleequipped with MMK1 membrane .The method demonstrated high efficacy in enterovirusconcentration from water with various degrees of contamination with the concentration time shortenedto 42 min. The MMK-type filtration membrane modifiedwith 0.5 % amino compounds proved most effective amongthe studied membranes. Hollow fiber MF membranes(d = 0.2 μm) were used to study the process of humanadenovirus 2 (HAdV-2) concentration depending on the virus content varying between 1.3 × 10To optimize the separation of hepatitis А virus fromwater, membranes based on polyamide, cellulose nitrate,cellulose acetate, and polyethersulfone were studied . Glass fiber, cardboard, andpolypropylenewere used as pre-filters. It was shown thatthe most effective membrane combination for filtration ofhighly impure water includes a cardboard or polypropylenefilter for preliminary filtration and a polyamide filter for the main stage of the process. A pressure filtration device was used for filtrationof samples with water volumes up to 10 L carried outat the pressure of 2 atm.Epidemiological surveillance over human enterovirusesrequires virus concentration from wastewater. This typicallyinvolves processing of large volumes (100–1000 L)of water, the most common method being the VIRADELprocess with microporous filters. Positively charged filtersdo not require sample pretreatment, and they can concentrateviruses from water with a wider pH range than electronegativefilters due to the virus surface charge typicallybeing negative. Virosorb 1MDS is one of the most commonelectropositive filters. In recent years, it has been joinedby positively charged NanoCeram filters . Virus elution from filters after concentrationwas performed using organic (beef extract) or inorganic(sodium polyphosphates) solutions. Then, the eluates wererepeatedly concentrated to reduce sample volumes andimprove virus detection. Most filters proved highly effectivein capturing the virus, while the elution and repeatedconcentration methods showed varying degrees of successdue to the biological variation of viruses present in water.Nanofiltration (NF) was used in the filtration of serumprotein solutions, production of serum derivatives, andmanufacturing of a wide range of biopharmaceutical products.Depending on the material used, NF membranes fallinto two main groups: ceramic and polymeric ones. HybridNF membranes are also of interest due to their performanceunder suboptimal conditions. Various polymers, such aspolyethersulfone, polydimethylsiloxane, polyvinylidene fluoride, and polysulfone were tested as top layer materials for hybrid membranes.The use of polymers for obtaining hybrid NF membranesmakes it possible to change pore sizes, chemical properties,and charges of membrane surfaces. Nanofiltrationincreasingly becomes a common step in pharmaceuticalproduction, since it does not raise toxicity issues . Obtaining a purified virus concentrate is oneof the key steps in vaccine production. Polyethersulfonemembrane 300 kD combined with weak anionic detergentsolution appears to be the most suitable for influenza virusconcentration and removal of small molecule impurities.Elution and repeated concentration methods were reportedin the literature . First, wastewaterwas concentrated with ViroCap cartridge filters, and thenCelite beef extract, ViroCap flat disc filters, InnovaPrepconcentrating pipettes, PEG/NaCl precipitation, andskimmed milk flocculation were used. PEG/NaCl sedimentationand skimmed milk flocculation turned out to bethe most effective methods among the five methods testedin experiments with poliovirus Type 1 (PV 1). Optimization of the skimmed milk flocculation method increasedPV 1 recovery nearly twofold as compared to PEG/NaClprecipitation.Tangential flow filtration is a common method of filtrationby size . Influenza viruswas used to test a two-step purification and concentrationprotocol for viral particles.Chromatography is a separation method based on the interactionbetween the target virus and column matrix. Ingeneral, separation functionality depends on charge, size,hydropathicity, and affinity.Ion exchange chromatography utilizes the phenomenonof ion exchange between an immobile solid phase and amobile liquid phase, which makes it possible to separatestructurally and morphologically similar viral particlesprovided they have different total charges and/or chargedistributions over their outer surfaces .Virus elution may require different salt concentrations, andthe method can be applied to any mixed infection if viruseshave different isoelectric points. A simple separation andconcentration technique for mixed infection by cowpeachlorotic mottle virus (CCMV) and cucumber mosaic virus(CMV) is proposed in .Monolithic supports made it possible to use chromatographyfor purification and concentration of various viruses. Unlike the classicalchromatographic supports with diffusion-based mass transferand relatively small pores, monoliths are characterizedby significantly enhanced mass transfer due to convectionand channel sizes of several microns. I. Gutiérrez-Aguirreet al. (2009) showed that CIM QA supports effectivelyretained rotaviruses present in stool samples, as well asin wastewater and river water samples. Concentration ofrotaviruses was achieved by elution of the retained viruseswith 1 М NaCl solution. The obtained viruses preservedtheir integrity, as confirmed by electron microscopy.Size exclusion chromatography (or gel filtration in caseof aqueous liquid phase) is a fluid chromatography based onvarying capabilities of different-sized molecules to infiltratethe pores of non-ionogenic gel acting as a stationary phase.In this separation method, a matrix of densely packed silicagel or agarose gel is used . The mainadvantages of this method are its simplicity and the lowcost of resins. Nevertheless, the method suffers from thelack of selectivity, requires low flow velocity, and showslow overall performance. Tick-borne encephalitis virus wasused to refine the gel filtration method for the Superdex 200column . The samples obtained weretested by the following methods: (1) the immunological(dot blot) technique for testing biological activity, (2) gasphaseelectrophoretic mobility macromolecular analysis(GEMMA) for determining particle sizes, and (3) atomicforce microscopy (AFM) or TEM for obtaining information on shapes and sizes of viral particles. The mean diameterof inactivated tick-borne encephalitis viral particles determinedusing GEMMA was 46.6 ± 0.5 nm, as opposed toAFM and TEM images, which showed the diameters ofabout 58 ± 4 and 52 ± 5 nm, respectively.Hydrophobic interaction chromatography is based onparticle adsorption at weakly hydrophobic surface at highsalt contents with subsequent elution with descending saltgradient. Virus surfaces include hydrophobic areas, whichare linked to immobilized hydrophobic ligands at chromatographiccarriers . The limitation ofthe method is illustrated by an example with adenovirus. It was found that large salt concentrationscould not only reduce the virus immunogenicitybut could also favor viral particle aggregation.Polyvinyl alcohol cryogel with pore sizes of 0.04–2.0 μmwas used in a concentration method with virus capturingby bioaffine sorbent . The use ofthe sorbent was tested in the concentration process for influenzaType А viruses H1N1 and H3N2, parainfluenzavirus type 6, smallpox virus, and FMD viruses. Bioaffinesorbent based on viscoelastic, non-brittle, and hydrolyticallystable PVA cryogel allows virus concentration notonly in a chromatographic column, but in reactors withsorbent mixing, which significantly intensifies mass transfer.The advantage of the method is that it provides thepossibility of concentration not only for small but also forthe largest (0.5 μm and above) viruses. According to theinvention description, the bioaffine sorbent may include notonly antibodies but also immobilized enzymes capable ofmodifying the virus.To monitor the presence of influenza virus in open waterbodies, a custom device was designed . It employed magnetic immunosorbents in whichantibodies were immobilized on a solid magnetic support.Sorption of influenza viruses onto polyaniline (PANI)and carbon nanotubes, as well as on PANI composites(nanotubes and grains) with or without silver, was studied. It was found that inclusion of silverincreased the sorption capability of PANI tubes in case ofallantoic influenza Type А viruses. PANI nanotube compositeswith 30 % silver appear to be the most promisingvirus sorption material in water solutions with regard tothe combination of properties.Hepatitis А virus (HAV) was used as a model for comparisonof various carbohydrate-binding lectins, includingconcanavalin А (Con A), wheat germ agglutinin, andsoybean agglutinin, based on their binding affinity to thevirus . Con A showed higher binding affinitythan other lectins. Con A-bound immunomagneticseparation combined with RT-PCR made it possible todetect HAV at 10–4 dilution of the initial virus concentration. It shows that Con A may be a promisingcandidate for HAV concentration.It was shown in several papers  that magnetic beads coated with anionicpolymers, such as poly- may be used for effective capture of varioustypes of viruses, which include human immunodeficiencyvirus Type 1 , respiratory-syncytialvirus , Borna disease virus , influenza virus , andDengue fever virus . The method waslater applied to adenovirus as well .Japanese researchers  worked on anew virus concentration method. They proposed and experimentallydemonstrated a virus concentration device,which employed the ion depletion zone generated by ionpolarization. The efficacy was assessed with fluorescentnanoparticles, Baculovirus, albumin, and dextran. Allsamples were successfully concentrated.A new virus concentration strategy using the expressionof human poliovirus receptor gene (hPVR) on Escherichiacoli cell surfaces was developed . The ability of modified bacterial cells to capture viralparticles was confirmed by TEM. This approach providesnew opportunities for effective capture and concentrationof water-transmitted viruses.To process field samples, unique microdevices capable ofeffective virus enrichment and concentration were designedusing carbon nanotubes with variable sizes (CNT-STEM). The intertube distance between CNTscould be set between 17 and 325 nm to precisely matchthe sizes of specific viruses. With this device, the authorsmanaged to identify two new strains.The paper recently published by B.N. Zajtsev et al.(2019) described the use of a Vivaspin centrifugal concentrator.Viral particles were concentrated on a double verticalpolyethersulfone membrane. Following centrifugation, themembrane was retrieved from the concentrator to assessthe number of particles sedimented on the membrane byEM examination of ultrathin sections.The desired research goal plays a major part in choosingthe virus concentration method. For instance, ecological,pharmaceutical, and medical problems require differentapproaches. For a water contamination test to be successful,one should filter hundreds of liters of water from aspecific area and then collect the virus material sedimentedon the surface and inside charged filters. This procedure istypically followed by the second filtration step using differentfilters with adjusted pore sizes .A series of gains have been achieved in designing materialsfor filtration membranes in recent years. However, muchis to be done for complete virus recovery from membranesand membrane surfaces.To improve the virus concentration technology, oneshould focus on surface interactions between viruses andfilter materials. In particular, effective elution methodsshould be developed to disrupt the bonds between filtermaterials and viruses to increase recovery. In diagnosticstudies in specific patients, the amount of material is oftenlimited to few milliliters. In many cases, especially whenit comes to mixed infections, electron microscopy turnsout to be one of the most reliable virus detection tools.Certainly, ultrathin sections can only be obtained at wellequippedEM laboratories, where EM diagnostics of virusinfections is a routine process.The authors declare no conflict of interest.Abbaszadegan M., Alum A., Abbaszadegan H., Stout V. Cell surfacedisplay of poliovirus receptor on Escherichia coli, a novelmethod for concentrating viral particles in water. Appl. Environ.Microbiol. 2011;77(15):5141-5148.Ali A., Roossinck M.J. A simple technique for separation of Cowpeachlorotic mottle virus from Cucumber mosaic virus in naturalmixed infections. J. Virol. Methods. 2008;153:163-167.Alonso M.C., Rodriquez J., Borrego J.J. Enumeration and isolationof viral particles from oligotrophic marine environments by tangentialflow filtration. Int. Microbiol. 1999;2(4):227-232.Bakhutashvili T.O., Gusev A.A., Dudnikov A.I., Mikhalishin V.V.,Shipilov V.I. A Method for Concentrating Viruses. Patent RF№ 1834289, 2002. (in Russian)Barth H.G., Jackson С., Boyes В.Е. Size exclusion chromatography.Anal. Chem. 1994;66(12):595-620.Beniac D.R., Siemens C.G., Wright C.J., Booth T.F. A filtrationbased technique for simultaneous SEM and TEM sample preparationfor the rapid detection of pathogens. Viruses. 2014;6:3458-3471. DOI 10.3390/v6093458.Burova E., Loffe Е. Chromatographic purification of recombinantadenoviral and adeno-associated viral vectors: methods and implications.Gene Ther. 2005;12:5-17.Dong H., Xiao K., Li X., Ren Y., Guo S. Preparation of PVDF/Al2O3 hybrid membrane via the sol-gel process and characterizationof the hybrid membrane. Desalin. Water Treat. 2013;51(19-21):3685-3690.Doodeji M.S., Zerafat M.M. A review on the applications of nanofiltration in virus removal and pharmaceutical industries. Glob.J. Nanomed. 2018;3(5):555624. DOI 10.19080/GJN.2018.03.555624.Falman J.C., Fagnant-Sperati C.S., Kossik A.L., Boyle D.S., MeschkeJ.S. Evaluation of secondary concentration methods forpoliovirus detection in wastewater. Food Environ. Virol. 2019;11(1):20-31.Flavigny E., Gaboyard M., Merel P., Fleury H. Magnetic particlemediatedvirus concentration for clinical virology. In: 104thGeneral Meeting of the American Society for Microbiology,New Orleans, American Society for Microbiology. May 22–27,2004. Washington, DC, 2004.Gentile M., Gelderblom H.R. Electron microscopy in rapid viraldiagnosis: an update. New Microbiol. 2014;37:403-422.Ghaee A., Zerafat M.M., Askari P., Sabbaghi S., Sadatnia B. Fabricationof polyamide thin-film nanocomposite membranes withenhanced surface charge for nitrate ion removal from water resources.Environ. Technol. 2017;38(6):772-781.Gias E., Nielsen S.U., Morgan L.A.F., Toms G.L. Purification ofhuman respiratory syncytial virus by ultracentrifugation iniodixanoldensity gradient. J. Virol. Methods. 2008;147(2):328-332.Goldsmith C.S., Miller S.E. Modern uses of electron microscopyfor detection of viruses. Сlin. Microbiol. Rev. 2009;552-563.Gutiérrez-Aguirre I., Banjac M., Steyer A., Poljsak-Prijatelj M.,Peterka M., Strancar A., Ravnikar M. Concentrating rotavirusesfrom water samples using monolithic chromatographic supports.J. Chromatogr. 2009;1216(13):2700-2704.Havlik M., Marchetti-Deschmann M., Friedbacher G., Messner P.,Winkler W., Perez-Burgos L., Tauer C., Allmaier C. Developmentof a bio-analytical strategy for characterization of vaccineparticles combining SEC and nanoES GEMMA. Analyst. 2014;139(6):1412-1419.Hazelton P.R., Gelderblom H.R. Electron microscopy for rapid diagnosisof infectious agents in emergent situations. Emerg. Infect.Dis. 2003;9:294-303.Ikner L.A., Gerba C., Bright K. Concentration and recovery of virusesfrom water: a comprehensive review. Food Environ. Virol.2012;4(2):41-67.Ismagambetov B.M., Koshemetov Zh.K., Bogdanova M.I., NakhanovaG.D., Nurabaev S.Sh., Seysenbaeva M.S., SansyzbaiA.R., Kasenov M.M. Design of diagnostic products forinfluenza A subtypes. Mezhdunarodnyy Zhurnal Prikladnykh iFundamental’nykhIssledovaniy. Seriya Biologicheskie Nauki =International Journal of Applied and Fundamental Research.Biological Series. 2017;10:260-264. (in Russian)Ivanova V.T., Ivanova M.V., Sapurina I.Yu., Burtseva E.I., TrushakovaS.V., Isaeva E.I., Kirillova E.S., Stepanova N.V., OskerkoT.A. A comparative study of carbon nanotubes and polymercomposites containing silver nanoparticles as sorbents of influenzaviruses A and B. Voprosy Virusologii = Problems of Virology.2015;60(3):25-30. (in Russian)Ko S.-M., Cho S.-Y., Oh M.-J., Vaidya B., Kim D. Application ofconcanavalin A-linked magnetic beads for the detection of hepatitisA virus. J. Food Prot. 2018;81(12):1997-2002.Krajacic M., Ravnikar M., Strancar A., Gutiérrez-Aguirre I. Applicationof monolithic chromatographic supports in virus research.Electrophoresis. 2017;38:22-23.Kyzin A.A., Zagidullin N.V., Gelich L.V., Timerbaeva R.H., IsrafilovA.G. Purification and concentration of influenza virus bymicro- and ultrafiltration. Vestnik Bashkirskogo Universiteta =Bulletin of Bashkir University. 2014;19(4):1223-1227. (in Russian)Levchenko I.V., Efremenko V.I., L’vov D.K., Zharnikova I.V.,Deryabin P.G., Vasilenko N.F., Isaeva E.I., Botikov A.V. Developmentof magneto-immunosorbent test systems and a devicefor selective concentration for the detection of avian influenzaviruses. Proceedings of the 9th Congress of the All-RussiaResearch and Practical Society of Epidemiologists, Microbiologists,and Parasitologists. Moscow, 2007;1:248-249. (in Russian)Lozinskiy V.I., Plieva F.M., Isaeva E.I., Zubov A.L. A Method ofConcentrating Virus. Patent, 2013. Available at: http://www.findpatent.ru/patent/213/2130069.html. (in Russian)Lu R., Li Q., Yin Z., Xagoraraki I., Tarabara V., Nguyen T. Effectof virus influent concentration on its removal by microfiltration:The case of human adenovirus 2. J. Membr. Sci. 2016;497:120-127.Maximous N., Nakhla G., Wan W., Wong K. Preparation, characterizationand performance of Al2O3/PES membrane for wastewaterfiltration. J. Membr. Sci. 2009;341(1-2):67-75.Mogi K., Hayashida K., Honda A., Yamamoto T. Development ofvirus concentration device by controlling ion depletion zone forultra-sensitive virus sensing. Trans. Sens. Micromachines. 2016;136(9):363-369.Reid G.G., Milne E.W., Coggins L.W., Wilson N.J., Smith K.T.,ShepherdA.J. Comparison of electron microscopic techniquesfor enumeration of endogenous retrovirus in mouse and Chinesehamster cell line used for production of biologics. J. Virol. Methods.2003;108:91-96.Roettger B.F., Myers J.A., Ladisch M.R., Regnier F.E. Аdsorptionphenomena in hydrophobic interaction chromatography. Biotechnol.Prog. 1989;5(3):79-88.Ruščić J., Gutiérrez-Aguirre I., Žnidarič M.T., Kolundžija S., SlanaA., Barut M., Ravnikar M., Krajačić M. A new application ofmonolithic supports: The separation of viruses from one another.J. Chromatogr. A. 2015;1388:69-78.Ryabinnikova A.I., Matraimov M.B., Shalgynbaev E.K., RystaevaR.A., Orynbaev M.B. Purification and concentration ofhorses rhinopneumonia virus. Nauka, Novye Tekhnologii i InnovatciiKyrgyzstana = Science, New Technologies, and Innovationsin Kyrgyzstan. 2015;4:129-131. (in Russian)Sakudo A., Baba K., Ikuta K. Capturing and concentrating adenovirususing magnetic anionic nanobeads. Int. J. Nanomedicine.2016;11:1847-1857.Sakudo A., Baba K., Tsukamoto M., Ikuta K. Use of anionic polymer,poly-coated beads forcapture of respiratory syncytial virus. Bioorg. Med. Chem. Lett.2009a;19(15):4488-4491.Sakudo A., Baba K., Tsukamoto M., Sugimoto A., Okada T., KobayashiT., Kawashita N., Takagi T., Ikuta K. Anionic polymer,poly-coated beads-basedcapture of human influenza A and B virus. Bioorg. Med. Chem.2009b;17(2):752-757.Sakudo A., Ikuta K. Efficient capture of infectious H5 avian influenzavirus utilizing magnetic beads coated with anionic polymer.Biochem. Biophys. Res. Commun. 2008;377(1):85-88.Sakudo A., Ikuta K. A technique for capturing broad subtypesand circulating recombinant forms of HIV-1 based on anionicpolymer-coated magnetic beads. Int. J. Mol. Med. 2012;30(2):437-442.Sakudo A., Masrinoul P., Tanaka Y., Ikuta K. Capture of denguevirus type 3 using anionic polymer-coated magnetic beads. Int.J. Mol. Med. 2011a;28(4):625-628.Sakudo A., Tanaka Y., Ikuta K. Capture of infectious borna diseasevirus using anionic polymer-coated magnetic beads. Neurosci.Lett. 2011b;494(3):237-239.Sanamyan A.G., Dmitrieva R.A., Doskina T.V., Lavrova D.V., NedachinA.E. Use of a membrane module MPM 0142 for the concentrationof viruses in the sanitary-virological surveillance ofwater objects. Gigiena i Sanitariya = Hygiene and Sanitation.2006;6:74-76. (in Russian)Schagen F.H.E., Rademaker H.J., Rabelink M., van Ormondt H.,Fallaux F.J., van der Eb A.J., Hoeben R.C. Ammonium sulphateprecipitation of recombinant adenovirus from culture medium:an easy method to increase the fetal virus yield. Gene Ther.2000;7(18):1570-1574.Segura M.M., Garnier А., Kamen А. Purification and characterizationof retrovirus vector particles by rate zonal ultracentrifugation.J. Virol. Methods. 2006;133(1):82-91.Sheets R.L. Opinion on adventitious agents testing for vaccines:Why do we worry so much about adventitious agents in vaccines?Vaccine. 2013;31(26):2791-2795.Soto-Beltran M., Ikner L.A., Bright K.R. Effectiveness of poliovirusconcentration and recovery from treated wastewater bytwo electropositive filter methods. Food Environ. Virol. 2013;5:91-96.Sun G., Xiao J., Wang H., Gong C., Pan U., Yan S., Wang Y. Efficientpurification and concentration of viruses from a large bodyof high turbidity seawater. MethodsX. 2014;1:197-206. DOI10.1016/j.mex.2014.09.001.Svec F., Perry G. Wang (Ed.). Monolithic chromatography andits modern applications. Anal. Bioanal. Chem. 2011;401:1459-1460. https://doi.org/10.1007/s00216-011-5175-0.Tarasov А.V., Fedotov Yu.А., Lepeshin S.А., Panov Yu.T., OkulovK.V., Vdovina А.I. The use of membranes with a positivesurface charge for sanitary and virological control of water. IzvestiyaSamarskogo Nauchnogo Tsentra Rossiyskoy AkademiiNauk = Proceedings of the Samara Research Center of the RussianAcademy of Sciences. 2012;14(1-9):2372-2376. (in Russian)Taylakova E.T., Chervyakova O.V., Sadikalieva S.O., SultankulovaK.T., Zaytsev V.L., Turganbaeva A.S., Sansyzbay A.R.Optimization of conditions for purification and concentration ofequine influenza virus. Vestnik Nauki KazATU imeni S. Sejfullina= Heraldof Science of the Saken Seifullin Kazakh Agro-Technical University. 2011;4(71):14-23. (in Russian)Transfiguracion J., Jorio H., Meghrous J., Jacob D., Kamen A. Highyield purification of functional baculovirus vectors by size exclusionchromatography. J. Virol. Methods. 2007;142(1-2):21-28.Trifonova E.A., Nikitin N.A., Kirpichnikov M.P., Karpova O.V.,Atabekov I.G. A method of production and characterization ofspherical particles, new biogenic platforms. Vestnik MoskovskogoUniversiteta. Seriya 16. Biology = Moscow UniversityBulletin. Series 16. Biology. 2015;4:46-50. (in Russian)Vicente T., Roldão A., Peixoto C., Carrondo M.J.T., Alves P.M.Large-scale production and purification of VLP-based vaccines.J. Invertebr. Pathol. 2011;107:42-48.Wickramasinghe S.R., Kalbfuss B., Zimmermann A., Thom V.,Reichl U. Tangential flow microfiltration and ultrafiltration forhuman influenza A virus concentration and purification. Biotechnol.Bioeng. 2005;92(2):199-208.Yeh Y.T., Tang Y., Sebastian A., Dasgupta A., Perea-Lopez N., AlbertI., Lu H., Terrones M., Zeng C.-Y. Tunable and label-freevirus enrichment for ultrasensitive virus detection using carbonnanotube arrays. Sci. Adv. 2016;2(10):e1601026. DOI 10.1126/sciadv.1601026.Yousefi M.H., Zerafat M.M., Shokri-Doodeji M.D., Sabbaghi S.Investigationof dip-coating parameters effect on the performanceof Alumina-Polydimethylsiloxane nanofiltration membranes for desalination.J. Water Environ. Nanotechnol. 2017;2(4):235-242.Zajtsev B.N., Taranov O.S., Rudometova N.B., Shcherbakova N.S.,Il’ichev А.А., Karpenko L.I. Optimized method for counting viralparticles using electron microscopy. Vavilovskii Zhurnal Genetikii Selektsii = Vavilov Journal of Genetics and Breeding.2019;23(3):237-242. DOI 10.18699/VJ19.498. (in Russian)Zajtsev V.P., Zolotykh D.S., Leonova V.D., Larskaya K.S., Krat I.P.,Orobinskaya V.N., Konovalov D.A. The nanoparticles: methodsof preparation and analysis, activity, and toxicity. SovremennayaNauka i Innovatsii = Modern Science and Innovation. 2016;3(15):197-218. (in Russian)Zalesskih A.A., Bystrova T.N. Improvement of the hepatitis A epidemiologicalsurveillance system based on the optimization of virologicaland serological monitoring. Meditsinskiy Al’manakh =Medical Almanac. 2018;4(55):70-74. DOI 10.21145/2499-9954-2018-4-70-74. (in Russian)


Lysiphlebus fabarum, a parasitoid of aphids. This virus, tentatively named “Lysiphlebus fabarum virus” (LysV), was discovered in transcriptome sequences of wasps from an experimental evolution study in which the parasitoids were allowed to adapt to aphid hosts (Aphis fabae) with or without resistance-conferring endosymbionts. Based on phylogenetic analyses of the viral RNA-dependent RNA polymerase (RdRp), LysV belongs to the Iflaviridae family in the order of the Picornavirales, with the closest known relatives all being parasitoid wasp-infecting viruses. We developed an endpoint PCR and a more sensitive qPCR assay to screen for LysV in field samples and laboratory lines. These screens verified the occurrence of LysV in wild parasitoids and identified the likely wild-source population for lab infections in Western Switzerland. Three viral haplotypes could be distinguished in wild populations, of which two were found in the laboratory. Both vertical and horizontal transmission of LysV were demonstrated experimentally, and repeated sampling of laboratory populations suggests that the virus can form persistent infections without obvious symptoms in infected wasps.We report on a novel RNA virus infecting the wasp  With thiscovery ,3,4,5,6.iscovery ,8 becausiscovery ,10,11,12iscovery ,14. ViraLysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae) is a parasitoid of aphids, in particular the black bean aphid, Aphis fabae (Hemiptera: Aphididae). Female wasps inject eggs into the aphid’s body [L. fabarum exist [L. fabarum is the most important parasitoid of A. fabae [A. fabae and contributes to the natural control of this pest aphid [Hamiltonella defensa, which increases the resistance of aphids, including A. fabae, against parasitoid wasps [H. defensa has been linked to the presence of a toxin-encoding bacteriophage called APSE in the H. defensa genome [Across all insects, a large number of species employ a parasitoid lifestyle, with up to 20% of all insects belonging to the parasitoid wasps ,16. As ad’s body , where tA. fabae . It impost aphid . Interesst aphid . The mosid wasps ,25. Prota genome ,27. ViraNasonia [Venturia [Dinocampus coccinellae [Dinocampus coccinellae paralysis virus (DcPV) in both wasp venom and in the neural tissue of infected ladybugs is associated with host paralysis behavior [While viruses are typically viewed solely as pathogens, their interactions with hosts can range from parasitism to commensalism, and even mutualism in insects ,29,30,31Lysiphlebus fabarum virus (LysV), isolated from the parasitoid wasp L. fabarum and bearing sequence similarities to DcPV. This virus was first identified in transcriptomic sequences obtained from experimentally evolved laboratory populations of the parasitoid L. fabarum [H. defensa, resulting in wasp populations with improved abilities to infect H. defensa–protected hosts [Here we present evidence for a previously unknown RNA virus, the  fabarum . These ped hosts ,36. FollH. defensa in aphid hosts [L. fabarum were reared on each of three different types of hosts: (1) a H. defensa-free clone of A. fabae , (2) a subline of the same aphid clone carrying a heritable infection with H. defensa strain H76 , and (3) a subline carrying H. defensa strain H402 . The study ran for 21 generations of experimental evolution, after which the four replicates of each treatment were merged into single populations of each type that continued to be reared on their respective hosts. We sampled wasps from these merged populations on two occasions, in April 2016 , and in October 2016 (57 generations). To disentangle a potential impact of H. defensa on viral reproduction, the two lines reared on H. defensa-infected aphids (H76 and H402) were also sampled following one generation of rearing in H. defensa-free aphids (H-). Sampled parasitoids were reared by placing five wasps of mixed sex on plants containing week-old aphids and collecting their offspring . We also screened the large, outbred sexual population that was used as the base population to found the experimental evolution lines in the study of Dennis et al. [We also screened for viral presence in laboratory stocks of sexually . Five ass et al. . This pos et al. . The popL. fabarum s. str. collected across Europe  during four separate sampling campaigns . First-strand cDNA was synthesized according to the manufacturer’s protocol . Viral presence/absence was determined using PCR, carried out with the first strand cDNA products using the GoTaq Green Master Mix  in 10 µL reactions and the following newly designed viral primers (5′ - 3′): LysV forward ATTTTCAGAGCTCCGTGGCA, LysV reverse CTGAAGCCCGAACAAAAACG. The thermal regime consisted of 15 min at 95 °C, with 27 subsequent cycles of the following three steps: 30 s at 94 °C, 1.5 min at 58 °C, 1 min at 72 °C followed by a final 30 min extension at 60 °C. PCR products were viewed on a 1.4% agarose gel stained with PeqGreen  and Sanger sequenced  to verify the target sequences.GAPDH), an enzyme involved in glycolysis and expected to be continuously transcribed. GAPDH showed no differential expression across the wasp lines and treatments used here [GAPDH was targeted with the following primers and probe (5′–3′): GAPDH forward ACTTGCCCTTCAAACGACAC, GAPDH reverse TCCACCACGTTCAAGGGATG, GAPDH probe ACGGTTTTGGACGTATTGGAC. The probes were modified with 5′ terminal reporter dyes  and 3′ terminal quenchers . All reactions were carried out in triplicate using 10 µL reaction volumes with 1 µL template cDNA on an ABI 7500 Fast Real Time PCR system . Viral expression was measured relative to the reference gene by creating standard curves (GAPDH RNA (linear range 1–10−3 ×), calculating primer efficiencies with the formula q analysis [A real–time quantitative PCR (qPCR) assay using a Taqman fluorescent probe was developed to quantify viral RNA in a subset of the individual wasps tested with our presence/absence PCR screen (above). All reactions were scaled to a reference gene: glyceraldehyde–3–phosphate dehydrogenase . To identify sequence variants within each individual library, we performed a second round of de novo assembly, this time using these 111 sequences as a reference for separate assemblies of the viral polyprotein from each of the 110 transcriptomic libraries, using the default genome-guided options in Trinity. We filtered the results of these independent assemblies to retain only the longest sequences (>5000 bp). Conserved protein domains were identified with a blastp search against the SwissProt database [LysV was initially detected from three transcripts that were highly differentially expressed between the experimentally-evolved populations of s et al. . We asses et al. ) and 4–5s et al. ). These 14.07.17 . Within 14.07.17 ) by blasdatabase .Picornavirales [The relationship of LysV relative to other known viruses was examined using a phylogeny based on the RdRP region, extracted from the two distinct viral polyproteins that were whole-genome assembled (see results). The highly conserved domains of the RdRP region have previously been used to construct informative phylogenies of RNA viruses ,58,59,60avirales ,61, inclavirales   under the same climatic conditions mentioned previously. All wasp lines in the transmission assay setup were reared on H. defensa free aphids of the genotype A08–28.In addition to viral presence across wasp lineages, it is also important to determine the mode of viral transmission . To test this, a transmission assay was set up with infected and uninfected wasp lines competing for the same aphid hosts, followed by rearing of their offspring . This enThe transmission assay was run over three generations . GeneratThree different types of wasp combinations were set up in the second generation. Assay type 1 contained female–female pairs (seven replicates) with only one infected H76 and one uninfected asexual female that were removed from the aphid hosts after 24 h. These female-female pairs were set up to trace the viral infection by direct screening through all three generations with knowledge of the specific mother-progeny combination. Type 2 assays contained male–female pairs (four replicates) with one infected male (H76) and uninfected female , and parental wasps were removed from the hosts after 24 h. The male–female pools completely excluded two possible modes of transmission , since males cannot inject eggs into aphid hosts. However, here it was possible that there is transmission by mating. The asexual females, although capable of reproducing asexually, may be forced to mate in combination with sexual males, possibly leading to viral transmission through the infected male. Assay type 3 contained larger pools of wasps (six replicates), with ≥4 of both infected (H76) and uninfected  individuals, and included some pools with only H76 females and some with H76 females and males . In the p < 0.05) with a mixed linear model (GAPDH (log transformed) for individual wasps served as the response variable. Wasp lines were considered separately and only lines with samples available for both sampled time points were compared. Unbalanced data was accounted for by using the Satterthwaite approximation for degrees of freedom. p-values of the fixed effect (generation) were calculated using the F–test and those of the random effect (sample within generation) using the likelihood ratio chi-square test. Samples of the transmission assay were tested for statistically significant differences in viral load also using a mixed model  between wasps sampled in April and October 2016 were tested for their statistical significance  showed viral presence  while no infected individuals were detected in the other two lines . Two of p-values ≤ 0.008), but not between time points   and at one time point for line IL07–64. No viral presence was ever observed in line IL06–242, used as the uninfected line in the transmission assays. Further, dissected wasp samples (ovaries and bodies) also demonstrated this higher sensitivity of the qPCR assay. Out of 18 dissected samples chosen for qPCR screening, eight additional samples tested positive for the virus, including one sample from the H– line, where no viral presence was previously found.With this more sensitive qPCR method, viral RNA was also found in three asexual wasp samples, previously tested negative with PCR. The virus was detected at both time points in line IL09–554 . To examine only the nearly complete polyproteins, we subsetted the 48 longest sequences (>5000 bp). Within these, there were two distinct LysV polyproteins, which we have labelled “A” and “B” to match with the Sanger sequencing described above . Among the 48 long sequences, LysV A and LysV B were equally represented . These two sequences differ by ca. 650 nucleotide variants  [Dinocampus coccinellae paralysis virus (DcPV) [Nasonia vitripennis virus isolate 1 (NvitV–1) [Both Bayesian inference and maximum likelihood analyses of the alignments of the RdRP region produced identical tree topologies and classified the RNA virus (haplotypes LysV A and LysV B) as belonging to the Iflaviridae family in the order of the Picornavirales . Its clo (VcPLV) , followes (DcPV)  and the NvitV–1) , all parn = 20), 50–100% of third-generation H76 offspring were infected.We observed evidence of both horizontal and vertical virus transmission, but horizontal transmission rates were much lower than vertical. Since it was most consistently infected, we were able to test and detect vertical transmission in all three assay types using the H76 line . Based oWe chose the previously uninfected IL06-242 line  to test As with vertical transmission, the horizontal transmission we detected was also imperfect. In each female-female pair, four of the five asexual wasps in generation 3 were virus positive (based on qPCR), and in the large pool assay only one individual was detected . ImportaL. fabarum revealed a previously undescribed species of RNA virus, which we have named Lysiphlebus fabarum virus (LysV). Prior to this study, blast results showed a close similarity of the LysV polyprotein to the polyprotein of DcPV [L. fabarum [Our analyses of RNA obtained from the aphid parasitoid  of DcPV , an RNA  of DcPV . Phyloge of DcPV ,73. The  fabarum , indicatL. fabarum, of which two were also present in the laboratory populations we studied. This diversity could be due to the rapid evolution rate that has been documented in viruses [L. fabarum, but it is very well possible that other aphid parasitoids carry LysV since other viruses, such as the Diadromus pulchellus ascovirus (DpAV), have been detected in more than one species belonging to the Ichneumonidae [Three different virus haplotypes were identified in field-collected  viruses ,76. The  viruses  only twoumonidae .Pteromalus puparum, an RNA virus leads to the deterioration of the venom apparatus [L. fabarum will also fall into one of these categories. It is also possible that further examination of virus-infected individuals will reveal that it has an influence on parasitoid success or behavior. The higher presence of LysV in experimentally evolved lineages with different infective abilities suggests that this virus could be advantageous in some settings [No obvious positive or negative effects have been observed in infected populations/individuals compared to non-infected ones. This points towards a persistent non-symptomatic infection of the virus, as has been reported for other viruses infecting parasitoids ,30,78. Fpparatus . These epparatus ,30, it isettings . HoweverThe transmission assays revealed that LysV can be both vertically transferred between mothers and offspring of the same lines as well as horizontally transmitted to individuals of different genotypes. Vertical transmission was more frequent than horizontal transmission, but both modes were imperfect. With mounting evidence of viruses infecting organisms with short generation times, such as arthropods, the importance of vertical transmission has been emphasized as a means of ensuring viral passage ,81,82,83L. boulardi [L. fabarum, as we have observed superparasitism in the lab [Distinguishing clearly between the different forms of transmission  can be difficult in systems such as these parasitoids because of their close proximity (in or on) their aphid hosts, and the possibility of multiple parasitoids attempting to infect the same host (superparasitism). Vertical transmission was observed in experimental setups containing only infected females, suggesting that the most likely form of vertical transmission is maternally through the eggs, but we could not distinguish this from viral deposition in the host followed by re-infection of the simultaneously injected egg. Maternal transmission through eggs has been observed in combination with horizontal transmission in a virus infecting the parasitoid wasp boulardi , blurrinboulardi ,69 while the lab . VisualiThe newly-infected asexual individuals obtained from the transmission assay all displayed a rather low viral load . This hints at a reduced viral load in newly-infected individuals, and the future outcome of these new infections needs to be investigated more thoroughly. High viral loads do not seem to be a requirement for transmission, however, as high fluctuations in viral loads across all generations of the transmission assay were observed: parental generations with very low viral loads could produce highly infected offspring and vice versa. Based on this, we conclude that any level of viral presence can lead to vertical transmission, but that not all offspring of infected parents carry the virus. If the high rates of vertical transmission failures we observed are representative of the natural situation, it would seem that they have to be compensated by relatively high rates of horizontal transmission or by some selective advantage of infected parasitoids for the virus to persist in host populations.


The coronavirus disease 2019 (Covid-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and presents a global health emergency that needs urgent intervention. Viruses constantly change through mutation, and new variants of a virus are expected to occur over time. In the United Kingdom (UK), a new variant called B.1.1.7 has emerged with an unusually large number of mutations. The aim of this study is to evaluate the level of protection of sera from 12 patients infected and later healed in Apulia Region  with Covid-19 between March and November 2020, when the English variant was not circulating in this territory yet, against the new VOC 202012/01 variant by seroneutralization assay. The sera of patients had already been tested before, using a virus belonging to the lineage B.1 and showed an antibody neutralizing titer ranging between 1:160 and 1:320. All the 12 sera donors confirmed the same titers of neutralizing antibodies obtained with a strain belonging to the lineage B.1.1.7 (VOC 202012/01). These data indicate that antibodies produced in subjects infected with variants of Sars-CoV-2 strain before the appearance of the English one, seem to have a neutralizing power also against this variant. Viruses, in particular RNA viruses, are often subject to mutations, and the appearance of new variants is a quite a common event. The SARS-CoV-2 variant virus that has emerged in the UK in December 2020, the so-called “English variant” VOC 202012/01), attracts particular attention due to its high contagiousness 2/01, att,4. This Preliminary reports by the United Kingdom indicate that this variant is more transmissible than previous circulating strains, with an estimated increase of between 40% and 70% in transmissibility .Laboratory studies are ongoing to determine whether these variant viruses have different biological properties or may alter vaccine efficacy .Currently, there is no evidence that these variants cause more severe illness or increased risk of death. On the other hand, the risk that substantial genomic alteration may lead to test failure or escape from antibody response or vaccine efficacy must be carefully considered, and new information about the virologic, epidemiologic, and clinical characteristics of this variants is rapidly emerging . As a maIn addition, convalescent plasma (hyperimmune plasma) is one of the most used therapeutic strategies to reduce mortality and severity of the disease in patients infected with Covid-19 [The aim of this study is to demonstrate that neutralizing antibodies produced by Italian patients who acquired the infection from March to November 2020, before the spread of VOC 202112/01, are fully protective also against this variant.Serum samples collected from 12 patients who acquired the Covid-19 infection between March 2020 and November 2020, were recruited as hyperimmune plasma donors for the Italian project TSUNAMI and used in the present study. All serum samples had been previously analysed by seroneutralization test using a strain isolated in Italy during the first stage of the pandemic, and all of them showed an antibody neutralizing titer ranging between 1:160 and 1:320.The lineage of the infecting virus was established for two patients, for whom the original diagnostic sample collected at the time of diagnosis could be retrospectively analysed by whole genome sequencing. For the other patients, for whom the infecting viral strain could not be retrospectively established, it is reasonable to assume that the strains were different from VOC 202012/01, since during the period in which the patients became infected, this variant did not circulate in the Apulia Region.The seroneutralization test was run in parallel with the VOC 202012/01 strain isolated in a biosafety level 3 (BSL-3) laboratory of the Istituto Zooprofilattico Sperimentale of Puglia and Basilicata , and with the virus isolate that has been used for the standardization of the test protocol that was established within the Italian network for SARS-CoV-2 seroneutralization test (NeuCoV-Net) established for the TSUNAMI protocol. The first one (GISAID accession number: EPI_ISL_745193), belonging to the lineage B.1.1.7 virus (VOC 202012/01), was isolated from a patient returning from Great Britain on 22nd of December 2020; the second one, belonging to the lineage B.1 clade G (GISAID accession number: EPI_ISL_568579), was isolated in March 2020 in Italy and was provided by Prof. Fausto Baldanti, Fondazione IRCCS Policlinico San Matteo .2 cell culture flask in Eagle’s minimal essential medium (EMEM)  supplemented with 10% (v/v) fetal bovine serum (FBS) , and 100 U/mL penicillin and streptomycin  in 5% CO2 at 37 °C.African green monkey kidney Vero E6 cells were used for both the propagation of SARS-CoV-2 and the neutralization assay. Cells were cultured into a 25 cmAll the procedures handling the SARS-CoV-2 and infected cell cultures were held in a BSL-3 laboratory. To produce the viral stock, the virus was propagated in Vero E6 cells, and culture medium was collected and centrifugate to remove the cell debris .50) end point method [4 Vero E6 cells (in 50 µL) were plated into 96-well plates. Then the stock solution of SARS-CoV-2 was diluted serially from 10−1 to 10−8, and 25 µL of each dilution was added to the cells and incubated in 5% CO2 at 37 °C for 72 h. Eight replicates were performed for each dilution and used to quantify the virus titer and statistically determine the TCID50 end point.Virus infectious titers were established by the Reed and Muench tissue culture infective dose  were added to each well. The results of the seroneutralization test were determined by the appearance after 72 h of CPE, which was observed under an inverted microscope Axiovert 25 . The neutralizing antibody titer was defined as the highest serum dilution at which no CPE breakthrough in any of the testing wells was observed.A cytopathic effect (CPE)-based micro-neutralization assay was conducted in 96-well microtiter plates . BrieflyThe occurrence of new variants in the evolution of Sars-CoV-2 is a natural event, but it raises concern as mutations may determine altered biologic characteristics of the virus, including increased contagiousness, as it is the case of VOC 202012/01, which appeared in the UK and spread to several countries. In fact, this variant harbors multiple mutations in the spike protein, improving the ability of the virus to bind and penetrate cells. One major concern is the possibility that a profoundly mutated spike protein may alter the recognition by neutralizing antibodies, raising concerns about the protective ability of the recently issued vaccines that are based on original spike protein sequences. The evidence provided in the present study, according to a recent report , indicatThe data of this study indicate that the antibodies produced in subjects infected with variants of Sars-CoV-2 strain circulating before the appearance of the VOC 202012/01 variant possess the same neutralizing power against this variant. Genomic surveillance aimed at performing an updated molecular map of circulating Sars-CoV-2 strains plays a fundamental role, since this virus can easily undergo genetic variations. We cannot exclude a priori that virus mutation(s) in the future might induce such a relevant structural change of the spike protein to require vaccine update and modification.