Wolbachia pipientis can reduce RNA virus replication and prevent transmission by Aedes aegypti, and also have the capacity to invade wild‐type populations, potentially reaching and maintaining high infection frequencies. Levels of virus transmission blocking are positively correlated with Wolbachia intracellular density. Despite reaching high densities in Ae. aegypti, transinfections of wAlbA, a strain native to Aedes albopictus, showed no blocking of Semliki Forest Virus in previous intrathoracic injection challenges. To further characterize wAlbA blocking in Ae. aegypti, adult females were intrathoracically challenged with Zika (ZIKV) and dengue viruses, and then fed a ZIKV‐containing bloodmeal. No blocking was observed with either virus when challenged by intrathoracic injection. However, when ZIKV was delivered orally, wAlbA‐infected females showed a significant reduction in viral replication and dissemination compared with uninfected controls, as well as a complete absence of virus in saliva. Although other Wolbachia strains have been shown to cause more robust viral blocking in Ae. aegypti, these findings demonstrate that, in principle, wAlbA could be used to reduce virus transmission in this species. Moreover, the results highlight the potential for underestimation of the strength of virus‐blocking when based on intrathoracic injection compared with more natural oral challenges.Transinfections of the maternally transmitted endosymbiont  wAlbA Wolbachia strain in Aedes aegypti blocks Zika virus transmission in saliva when virus is delivered orally via an infectious bloodmeal.A transinfection of the wAlbA was not observed when virus was delivered via intrathoracic injection, suggesting that the midgut barrier plays an important role in blocking.Significant Zika blocking by Intrathoracic challenges can misrepresent virus blocking strength and may underestimate the true blocking potential of a strain. Wolbachia are maternally‐transmitted alphaproteobacteria widespread among the phylum Arthropoda. These endosymbionts are obligately intracellular, comprising a large number of distinct strains distributed among a wide diversity of host species. Wolbachia strains are currently classified into a set of 16 phylogenetically distinct supergroups (A–Q)   is a sperm modification that results in sterility unless a compensatory Wolbachia rescue factor is present in the embryo. The coupling of CI rescue with maternal transmission generates a relative reproductive advantage for Wolbachia‐infected females, with frequency thresholds for population invasion largely determined by the balance between the fitness benefits of CI and any negative effects on life history  and wAlbB (supergroup B) Wolbachia strains, where wAlbA tends to exist at a low intracellular density relative to wAlbB  hand‐held microinjector. Injected mosquitoes were immediately transferred to an incubator set to 27 °C for recovery. DENV injected females were left for 10 days prior to RNA extraction and virus quantification by a quantitative reverse transcriptase‐polymerase chain reaction. ZIKV injected females were left for 7 days. DENV was serotype 2, New Guinea C strain, obtained from Public Health England culture collections. The concentration of injected DENV virus was 2.5 × 108 FFU/mL. ZIKV was strain MP1751, obtained from Public Health England culture collections. The concentration of injected ZIKV virus was 4.8 × 108 FFU/mL. The primers used to measure DENV genome copies were DENV‐NS5‐F: 5′‐ACAAGTCGAACAACCTGGTCCAT‐3′ and DENV‐NS5‐R: 5′‐GCCGCACCATTGGTCTTCT‐3. The primers used to measure ZIKV genome copies were ZIKV‐835: 5′‐TTGGTCATGATACTGCTGATTG‐3′ and ZIKV‐911c: 5′‐CCTTCCACAAAGTCCCTATTGC‐3′.For the intrathoracic challenges, 30 5‐day old female mosquitoes from the wAlbA and wild‐type females were fed an infectious blood‐meal containing 1.4 mL of washed rabbit erythrocytes and 700 μL of viral suspension supplemented with ATP at a final concentration of 5 mm. The day before the infectious blood‐meal, batches of 65 females were isolated in feeding boxes and starved for 24 h. Mosquitoes were then exposed to the ZIKV NC‐2014‐5132 strain containing a final viral titre of 107 TCID50/mL.For the oral infections, 7‐day‐old wAlbA and 30 wild‐type mosquitoes were analysed at days 4, 7, 11, 14 and 21 post infection. To estimate infection, dissemination and transmission, mosquito bodies (thorax and abdomen), heads and saliva were analysed, respectively. To assess the transmission rate and transmission efficiency, mosquito saliva was collected from individual mosquitoes. Infection rate was determined by the proportion of mosquitoes with infected body (abdomen and thorax) among tested mosquitoes. The disseminated infection rate corresponds to the proportion of mosquitoes with infected heads among the previously detected infected mosquitoes (i.e. abdomen/thorax positive). The transmission rate refers to the proportion of mosquitoes with infectious saliva among mosquitoes with disseminated infection. Virus was titrated by plaque assay.For the infection, dissemination and transmission analysis, population batches of 30 wAlbA strain to inhibit ZIKV and DENV in Ae. aegypti was assessed via virus intrathoracic injection and was compared with the blocking capacity of wAu, a strain with comparable intracellular densities but that had previously shown strong viral inhibition . The wAu‐carrying line showed significantly reduced levels of both viruses , with 16 out of 24 (66.7%) ZIKV injected females containing no detectable virus compared with 100% virus positivity in wild‐type and wAlbA‐infected mosquitoes , whereas wild‐type females were capable of transmitting infectious virus after 14 days post infectious blood meal .To assess the blocking potential of the Wolbachia‐mediated virus transmission blocking. Although present at lower densities than in salivary gland or ovary tissues, wAlbA is found in the cells of the Ae. aegypti midgut epithelium (Ant et al., wAlbA line suggests that, even at a relatively low level, Wolbachia can limit the ability of ZIKV to cross the midgut barrier and escape into the haemolymph. Intrathoracic inoculation artificially bypasses the midgut cells, introducing high viral titres directly into the haemocoel. The data reported in the present study therefore highlight the importance of conducting oral challenges when assessing the potential of different Wolbachia strains as arbovirus control agents. Although technically easier to conduct, intrathoracic virus challenges alone can produce misleading results with respect to transmission‐blocking potential. Moreover, these findings illustrate the greater resolution achieved when virus blocking is characterized in terms of head dissemination, as well as virus titres in saliva, compared with whole‐body quantification. wAlbA can now be added to the small but growing list of Wolbachia strains that have been demonstrated to block transmission of ZIKV in Ae. aegypti and also have potential as tools for use in arbovirus control. Given the variability in blocking capacity and considering that comparative fitness effects can vary under different environmental conditions such as ambient temperature (Ulrich et al., et al., et al., Ae. aegypti transinfections with as many strains and therefore phenotypes as possible.The difference in levels of virus inhibition between the intrathoracic and oral challenges highlights the biologically crucial role of the midgut and salivary gland barriers in


Nowadays, male infertility is regarded as a global problem.Male infertility occurs due to abnormal sperm productionor function that prevent the delivery and/or the qualityof sperm. Infections, chronic illnesses, lifestyle choices andother factors can play a role in male infertility , 2.Most of viral infections indeed, are able to affect boththe reproductive tract tissue and the semen of humans andanimals, impairing sperm parameters and DNA integrityby different pathogenetic mechanisms that are responsiblefor temporary or permanent infertility in males andfemales .Considering the well-known evidence on relations betweenhuman immunodeficiency virus (HIV), humanpapilloma virus (HPV), cytomegalovirus (CMV), adenoviruses,parvovirus, mumps and fertility, the real questionis “Can the SARS-CoV-2 pandemia possibly influencemale patency?” Coronaviruses are enveloped, positivesingle-stranded large RNA viruses that infect both animalsand humans .The first example is the avian infectious bronchitis virus(AIBV), a coronavirus known to cause an acute respiratoryinfection in chickens. In addition to that, AIBV causesthe formation of epididymal stones in roosters, withdetrimental effects on sperm production and fertility. Thepresence of such calcium carbonate stones in the efferentduct of roosters causes the erosion of the epithelial lining,the reduction of testis weight and impairment of spermproduction lowering the fertility for 35-40%.Secondly, porcine reproductive and respiratory syndromevirus (PRRSV), a small enveloped RNA virus,shows a negative impact on fertility. In fact, the PRRSVprimarily attacks pulmonary alveoli to further replicate inepithelial germ cells (mainly spermatids and spermatocytes)inducing apoptosis and increasing the number ofimmature sperm cells. The overall effect is a diminishedquality of the semen. Moreover, this kind of replication permits sexual transmission of the virus that could be easilyidentified in ejaculate.Thirdly, the blue tongue virus (BTV) - a double strandedRNA virus that affects rams – represent another exampleof viral infection-caused semen impairment. The BTVRNA was detected in 75-100% of semen samples obtained25-57 days post clinical signs with notable impact on semenmotility, concentration and vitality. Although verylittle is known regarding the pathogenic mechanisms, theincreasing number of germ cell at different maturationstages and early signs of germinal epithelial regenerationsuggest a previous severe degeneration and sloughing ofgerm cells.Recent evidence reports that SARS coronavirus could also lead to a fertility damage, despitethe absence of a direct mumps-like orchitis. It was hypothesized that SARS coronavirus mayutilize the ACE-2 receptor expressed on testicular tissue. SARS patients’ testes displayedindeed a widespread germ cell destruction with thickened basement membrane and leukocyteinfiltration, mainly macrophages, compatible with findings of previous animal studies . Thepre


Nonresponding pneumonia is responsible for the most mortality of community-acquired pneumonia (CAP). However, thus far, it is not clear whether viral infection plays an important role in the etiology of nonresponding CAP and whether there is a significant difference in the clinical characteristics between viral and nonviral nonresponding CAP.  From 2016 to 2019, nonresponding CAP patients were retrospectively enrolled in our study. All patients received bronchoalveolar lavage (BAL) and virus detection in BAL fluid by multiplex real-time polymerase chain reaction (PCR), and clinical, laboratory, and radiographic data were collected. n = 8) and adenovirus (n = 7) were more frequently detected, and others included herpes simplex virus, human enterovirus, cytomegalovirus, human coronavirus 229E, rhinovirus, and parainfluenza virus. Compared with nonviral nonresponding CAP, only ground-glass opacity combined with consolidation was a more common imaging manifestation in viral nonresponding CAP. However, no obvious differences were found in clinical and laboratory findings between the presence and the absence of viral infections.  A total of 43 patients were included. The median age was 62 years, and 65.1% of patients were male. Overall, 20 patients (46.5%) were identified with viral infection. Of these viruses, influenza virus ( Viral infections were particularly frequent in adults with nonresponding CAP. The ground-glass opacity combined with consolidation was a specific imaging manifestation for viral nonresponding CAP, while the clinical and laboratory data showed no obvious differences between viral and nonviral nonresponding CAP. In 2016, lower respiratory infections caused more than two million deaths in people of all ages all over the world .Streptococcus pneumoniae was the most common pathogen [The response of CAP may be inadequate despite antimicrobial treatment, resulting in worsening of the symptoms that may lead to the spreading of infection, development of complications, and even death, which was named nonresponding pneumonia , 6. In tpathogen , 7. But pathogen . It has pathogen . A studypathogen . Howeverpathogen , 14, and9/L) were excluded. The study was approved by the Institutional Review Board for Human Studies of Second Affiliated Hospital of Zhejiang University School of Medicine .From January 2016 to January 2019, all adults (≥18 years of age) who had been admitted to the Second Affiliated hospital of Zhejiang University School of Medicine in China for CAP, underwent BAL, and were tested for respiratory viruses for poor response to empiric antibiotics therapy were enrolled in the retrospective study. Patients with severe immunosuppressive  and/or clinical symptoms after 72 hours of antibiotic treatment, chest radiographic progression of pneumonia , empyema, septic shock, and/or the need for mechanical ventilation.Clinical, radiographic, and laboratory data and short-term outcomes were collected by a trained physician. A bronchoscope examination was implemented for every patient after admitted to the ward of respiratory department for more than 24 hours later. And BAL fluid was collected from a predominant lesion area following a standardized protocol.Microbiological studies included the following: sputum, BAL fluid for Gram staining and culture; BAL fluid for virus detecting by multiplex real-time PCR, consisting of influenza virus A and B, respiratory syncytial virus A and B, adenovirus, human metapneumovirus, parainfluenza virus types 1 to 4, enterovirus, rhinovirus, cytomegalovirus, herpes simplex virus, human coronavirus 229E/NL63, human coronavirus OC43, human coronavirus HKU1, and bocavirus by using a multiplex real-time PCR kit . The performance of the respiratory virus multiplex real-time PCR kit used in this study has been evaluated in our previous published studies .A diagnosis of respiratory viral infection was made if a virus was detected by BAL fluid. A diagnosis of bacterial infection was made if a viral pathogen was not detected, and the following criteria were met: isolation of a respiratory pathogen from purulent sputum , BAL fluid, or blood culture. A mixed infection was defined as the presence of both respiratory virus and bacteria, as defined above. Lastly, if no pathogens were detected, based on the tests used in the study protocol, we classified this as “unknown.”χ2 test and Fisher exact test were used regarding categorical variables, while Student's t-test and Mann–Whitney U test were applied in continuous variables, as appropriate. All data were analyzed using a statistical software package .Categorical variables were described using frequencies and percentages, and continuous variables were presented as means and standard deviation or as median and interquartile range for data. The Forty-three of nonresponding CAP patients were sampled in this study shown in , with a Forty-three of BAL fluid specimens were obtained from nonresponding CAP patients. 29 patients (67.4%) had respiratory virus or bacteria identified, of which 20 patients (46.5%) had viral infection and 18 patients had bacterial infection shown in . Nine pan = 8) and adenovirus (n = 7). Others contained herpes simplex virus, human enterovirus, cytomegalovirus, human coronavirus 229E, rhinovirus, and parainfluenza virus. Two patients had two viruses isolated: one had human enterovirus plus adenovirus and the other had cytomegalovirus plus adenovirus. And another patient had human enterovirus, adenovirus plus coronavirus 229E detected.Among 20 patients with viral infection, the most common microorganisms were influenza A virus (As shown in n = 20), ground-glass opacity combined with consolidation (n = 14), and simply ground-glass (n = 13) were also the common HRCT findings among nonresponding pneumonia. Others like peribronchial lesion, nodule, and atelectasis could also be detected. Compared with nonviral nonresponding pneumonia, only ground-glass opacity combined with consolidation was a more common imaging manifestation in viral nonresponding pneumonia (P < 0.05), while others showed no differences. These data suggested that the ground-glass opacity combined with consolidation was a specific imaging manifestation for viral nonresponding pneumonia.All 43 patients were examined with high resolution computed tomography (HRCT) scan. As shown in The average hospitalization period was 16.9 days. There was high mortality (41.9%) for this cohort of nonresponding CAP patients, which was consistent with the fact that the cure rate of nonresponding CAP was low, while the mortality rate showed no significant difference between the viral and nonviral nonresponding pneumonia.Our study provides three important findings: (1) the viral infections were particularly common in patients with nonresponding CAP; (2) the ground-glass opacity combined with consolidation on HRCT images was a specific imaging manifestation for viral nonresponding CAP; (3) the clinical and laboratory data showed no obvious differences between viral and nonviral nonresponding CAP patients.In previous studies, the incidence of nonresponsiveness in hospitalized CAP was 10% to 15% . And we Legionella, Staphylococcus aureus, or Gram-negative bacteria prolongs hospitalization period and is more likely to result in death [Several factors like the causal microorganisms, the host, and the antibiotics are relevant with nonresponding pneumonia, and to a large extent the reaction to antibiotics depends on the infection factors. Bacteria is a major component of pathogen causing CAP. Pneumonia caused by in death , 20. Howin death  found thin death  also fouViral pneumonia is most typical in children, but with advanced technology, more and more adults are identified with viral infection. In adults, the viral infection rate was found about 10–23% in CAP. Some studies even noted that a third of cases were related with viral infection. The percentage increased more when real-time PCR was utilized. Of these cases, influenza virus, rhinovirus, and coronavirus were relatively frequently detected , 23, whiIn this study, there was a total increase in leukocytes, neutrophils, CRP, and PCT. And a reduction in blood oxygen partial pressure, oxygen saturation as well as oxygenation index could be detected oppositely, demonstrating that patients were far from reaching a stable state. In nonresponding pneumonia, there was a persistent lung and systemic inflammation. Leukocytes and neutrophils (including both percentage and absolute number) in blood were significantly higher than normal, representing the regulation of inflammation for killing microorganisms, which could modulate inflammatory cytokine response . And in Unfortunately, we found no significant differences in serum markers and clinical features between two groups whether infected virus or not. Undoubtedly, symptoms and laboratory and radiological data are only a supplemental tool to support the current clinical state. Pathogenic identification, after all, is golden standard and important basis for effective treatment methods. The samples can be taken by either invasive or noninvasive ways. Sputum, nasopharyngeal/oropharyngeal swabs, and blood samples are common noninvasive detection methods to collect samples for an etiological diagnosis of CAP . HoweverThere are some limitations in this study. First, it is a retrospective study so that the data are limited. Some clinical data like oxygenation index are not available in several nonresponders for the loss of arterial blood gas analysis, and selection bias also follows. Second, the deficient number of cases affects the credibility of the result. Finally, the actual incidence of bacterial etiology might be underestimated for almost all people have received prior antibiotic treatment.In conclusion, the proportion of respiratory virus infection is high among patients with nonresponding CAP, and in patients with nonresponding pneumonia, it is difficult to distinguish patients with viral infection or not. Multiplex real-time PCR in BAL fluid is an effective diagnosis approach for respiratory virus identification even when antibiotic therapy has been performed. In terms of treatment, for simple viral nonresponding pneumonia, targeted antiviral treatment should be selected instead of abuse of antibiotics.


In 2018, a strain of epizootic hemorrhagic disease virus (EHDV), named YNDH/V079/2018, was isolated from a sentinel calf in Mangshi County, Yunnan Province, China. Nucleotide sequencing and neutralization tests indicated that the virus belongs to a novel serotype of EHDV that had not been reported previously. Reoviridae, genus Orbivirus) is transmitted between ruminants by Culicoides spp. biting midges. It is widespread in tropical and subtropical regions and primarily infects white-tailed deer and cattle  and real-time quantitative reverse transcription PCR (qRT-PCR)  of Seg-2 and Seg-3 sequences (GenBank accession nos. MN418446 and MN418447) revealed the highest matching identities with equivalent genome segments of other EHDVs.Double-stranded RNA extracted from YNDH/V079/2018 generated a genome segment migration pattern typical of bluetongue virus or EHDV  Figure 2rbivirus  Figure 3Subcore shell viral protein (VP) 3 of EHDV, encoded by Seg-3, is highly conserved, showing >95.5% amino acid sequence identity within EHDVs , panel AThe outer capsid protein VP2 of EHDV, encoded by Seg-2, is highly variable and is definitive for serotype determination , panel BWe developed conventional RT-PCR and qRT-PCR targeting Seg-2 of YNDH/V079/2018  Table 1 To determine the prevalence of YNDH/V079/2018, we tested 87 EDTA cattle blood samples from 3 farms in Mangshi County during April–October 2018 but detected no nucleic acid consistent with YNDH/V079/2018. Additional genome sequencing, type-specific diagnostic tests, and epidemiologic and pathogenic investigations of this novel EHDV are planned. Our study will help clarify the diversity of EHDV serotypes and the distribution and pathogenicity of this novel EHDV and its potential risk to ruminants.Additional information about epizootic hemorrhagic disease virus, China.


Lily mottle virus (LMoV), Tulip breaking virus (TBV) and Rembrandt tulip-breaking virus (ReTBV). Recombination prediction accomplished with Recombination Detection Program (RDP) v4.98 revealed potential intraspecies recombination in the case of TBV and LMoV. Phylogenetic analyses of the coat protein (CP) regions proved the monophyletic origin of these viruses and verified them as three different species according to current International Committee on Taxonomy of Viruses (ICTV) species demarcation criteria. Based on these results, we analyzed taxonomic relations concerning potyviruses associated with tulip breaking syndrome. We propose the elevation of ReTBV to species level, and emergence of two new subgroups in ReTBV.Tulip breaking is economically the most important viral disease of modern-day tulip growing. It is characterized by irregular flame and feather-like patterns in the flowers and mosaic on the foliage. Thirty-two leaf samples were collected from cultivated tulip plants showing tulip breaking syndrome from Hungary in 2017 and 2018. Virus identification was performed by serological (ELISA) and molecular (RT-PCR) methods. All samples proved to be infected with a potyvirus and evidence was provided that three potyvirus species could be identified in the samples:  Tulipa [Tulipa gesneriana [Tulips, as ornamental plants, have first been described as early as 1576 by Carolus Clusius [Tulipa ,3. CurreTulipa  with morsneriana ,3,6. Tulsneriana ,9, and isneriana ,9. The vsneriana . Later, sneriana . Tulip bsneriana . Symptomsneriana ,13,14. Isneriana . InfecteSeveral species of viruses were identified on tulips from different genera like Arabis mosaic virus (Nepovirus), Lily symptomless virus (Carlavirus), Cucumber mosaic virus (Cucumovirus), Potato virus X and Tulip virus X (Potexvirus), Tobacco necrosis virus (Necrovirus), Tobacco rattle virus (Tobravirus), and different members of the family Potyviridae, genus Potyvirus ,14. The Previously different potyviruses were identified from tulips, including tulip breaking virus (TBV), tulip band-breaking virus (TBBV), tulip top-breaking virus (TTBV) that is a strain of turnip mosaic virus (TuMV-TTB), Rembrandt tulip-breaking virus (ReTBV), and lily mottle virus (LMoV) .Lily mottle virus : AJ564636, RefSeq: NC_005288) [According to the International Committee on Taxonomy of Viruses (ICTV) four tulip infecting potyvirus species are accepted. _005288) ,18 was a_005288) . Tulip b_005288) , and it _005288) ,22. The Tulip breaking virus  [_043168) ,23 was a_043168) , the fir_043168) . It is pTulip mosaic virus  [_043425)  was iden_043425) , but theTurnip mosaic virus  [Tulip chlorotic blotch virus ,23 and T_002509)  and occu_002509) . Rembrandt tulip-breaking virus  was accepted as species in 1995 , but movAs data on nucleic acid sequence accumulated, the virus species demarcation got stricter and a lot of inconsistencies have been cleared up. In the case of potyviruses ICTV species demarcation criteria requires less than 82% identity of amino acid and less than 76% identity of nucleotide sequence of the polyprotein or CP  and adeqIn the present study, we aimed to determine the diversity of potyviruses present in Hungarian tulip plants showing typical color breaking syndrome. We also analyzed the taxonomic relationships between tulip infecting potyviruses and incidence of recombination in the evolution of tulip infecting potyviruses.Thirty-two tulip samples showing typical tulip breaking symptoms were collected in different locations in Hungary .All of the collected tulip samples proved to be infected with a potyvirus, according to potyvirus specific ELISA tests . The nucAll virus infected plants showed early symptoms, purple-burgundy stripes, or flecks on the abaxial surface of the leaves early in the season A,C, but Recombination Detection Program (RDP) v4.98 predicted six recombination events with at least 3 different methods . Each evp-values (Bonferroni corrected) were calculated for each recombination event, the highest acceptable p-value was 0.05  multiple recombination events were identified. In the case of TBV isolates three potential recombination events were predicted, but no evidence of potential recombination was found in ReTBV isolates. Most of the predicted recombination breakpoints are located in the CP, but recombination points in the NIb protein coding region and in the 3′ UTR were also predicted .Lilium longiflorum two recombination events were predicted and one recombination was predicted in a Lilium strain of LMoV (acc. #: AJ310203).Three potential recombination events were predicted in the case of LMoV. In an Australian strain (acc. #: JN127341) isolated from In the case of the Australian LMoV strain (acc. #: JN127341), in the first predicted recombination, the major parent is unknown, the program used MK368802 (subgroup I) to infer the potential major parent, minor parent is the LMoV strain from Ohio . The first breakpoint is at position 645 at the amino-terminal part of the CP, ending breakpoint is undetermined, but probably at position 1398, also located in the CP. Graphic representation of the event and Neighbour Joining (NJ) trees of both parents are shown on In case of the other predicted recombination in the LMoV strain acc. #: JN127341 (5th recombination event) the major parent was identified as LMoV acc. #: AJ310203 (subgroup I)—which was detected as a recombinant in event four—potential minor parent is unknown, the program used LMoV strain MK368802 (subgroup I) to infer it. The beginning breakpoint was at position 78 in the NIb protein coding region, while the second breakpoint was around position 644 in the amino-terminal region of the CP. In this sequence, the program was unable to identify sharp breakpoints as it was overridden due to multiple recombination events. Graphic representation of the event and NJ trees of both parents clearly showed that the fragment of the recombinant strain corresponding to the minor partner has a distinct position on the phylogenetic tree . AccordiIn the 4th predicted recombination event, an LMoV strain from China  was identified. The major parent was a LMoV strain described as TBV-lily strain  S44147,. This clJN127341 is potentially recombinant of subgroups I and II (event 1 and 5) and also indirectly recombinant of both subgroups by having AJ310203 as a major parent in event 5 . The parTulip mosaic virus) from Japan (acc. #: X63630) and two from the Netherlands (acc. #: KF440423 and KT923168).Three possible recombinants were predicted in the case of TBV; one in a TBV strain (currently accepted as In the case of the TBV isolate derived from Japan (2nd recombination event) (acc. #: X63630) the major parent is a Hungarian TBV isolate (MK368783) and potential minor parent is a recombinant strain from the Netherlands (KF442403). The first breakpoint is undetermined, probably at position 252, but in this sequence only the CP region was available, ending breakpoint is at position 1321 in the CP coding region. Interestingly the minor parent (acc. # KF442403) is marked as recombinant in event 3, this means X63630 is recombinant of a recombinant sequence. The graphic representation of the event and NJ trees of both parents clearly demonstrates that the fragment derived from the major and from the minor partner has distinct location , correspThe third predicted recombination event was in the 3′ UTR region of a TBV isolate derived from the Netherlands (acc. #: KF442403) In this case the major parent was a TBV strain from the present study (acc. # MK368787) and the potential minor parent is unknown, the program inferred MF983710 as minor parent. The breakpoint is at position 1598. Graphic representation of the event and NJ trees of both parents demonstrate the distinct origin of the fragments .In the case of the other recombinant strain derived from the Netherlands  the major parent is unknown, the program inferred MK368783 as major parent, potential minor parent is a TBV strain from Hungary (MK368785). The breakpoint is at position 1004. Graphic representation of the event and NJ trees of both parents and the fragments of the recombinant strain clearly demonstrates the distinct origin. The amino-terminal region of the recombinant grouped together with the major parent and the carboxy-terminal region grouped with the minor parent . Phylogenetic analysis of the 32 CP sequences determined in this study and 18 CP sequences available in the GenBank database were carried out. For CP sequences the Tamura–Nei nucleotide substitution model  with GamAccording to the phylogenetic tree the three viruses, TBV, LMoV, and ReTBV have clearly monophyletic origin, but are distinct species. TBV is located in a distinct clade while LMoV and ReTBV strains formed a monophyletic group supported by high bootstrap values (100%). In this monophyletic group the LMoV and the ReTBV strains formed their own monophyletic group. Both the LMoV and ReTBV isolates formed two distinct subgroups according to the high bootstrap values (100%). The two subgroups (“Tulip breaking virus lily strain” and “Tulip band breaking virus”) of LMoV were described previously by Zheng et al. . HoweverLilium longiflorum cv. Flevo (acc. #: S44147) was described as TBV Lily strain [All of the isolates of subgroup II originated from different lily species and this subgroup has a worldwide distribution since it was identified in different locations, such as Australia, New Zealand, Japan, China, India, USA, and the Netherlands. One of these isolated in the Netherlands from y strain , but accThe TBV isolates form a much more compact clade and contains all of the isolates derived from tulip from the Netherlands, Japan and Hungary.The phylogenetic networks based on the complete nt sequences of the clones including the carboxy-terminal region of the NIb, the CP coding region and the 3′ end non-coding region  also supNucleotide (nt) sequence similarities of the complete CP regions of isolates used in the Maximum Likelihood (ML) phylogenetic tree were analyzed in detail. Nucleotide identities between the LMoV strains were at least 85.89%, in the case of TBV strains 94.78% while in the ReTBV isolates at least 89.38% identity have been found . Among tMoreover, amino acid (aa) sequence similarities of the complete CP regions were analyzed. In the case among the LMoV strains, at least 87.23% identity, in the case of TBV strains, 97.01%, while in the ReTBV isolates, at least 95.49% identity have been found . Among tThe NIb/CP polyprotein cleavage sites were also identified, where possible. In the case of LMoV the cleavage site was VAFQ/A in all of the 21 isolates. In all of the 19 TBV isolates, VQFQ/A was the cleavage site. Among ReTBV isolates, five sequences showed VIFQ/A, while in one case VILQ/A was identified as cleavage site . AccordiIn this study, we identified and characterized tulip infecting potyviruses based on samples collected in Hungary. All of the collected tulip plants with characteristic tulip breaking symptoms were proved to be infected with potyviruses, three distinct species were identified: LMoV, TBV, and ReTBV. Evidence was provided that MAb PTY1 antibody is able to detect all of these tulip infecting potyviruses. According to the symptoms of infected cultivars and the causative agents, we could not link a specific symptom type to a virus or vice versa. The ReTBV was identified exclusively in the Rembrandt group of tulips. In the case of TBV and LMoV, we could not confirm host preference towards a cultivar group or cultivar. This finding contradicts current knowledge as Darwin-hybrids are thought to be not susceptible to TBV ,13. No dWatermelon mosaic virus [Narcissus viruses [Bean yellow mosaic virus [Canna yellow streak virus [Lettuce mosaic virus [Pea seed-borne mosaic virus [Plum pox virus [Potato virus Y [Turnip mosaic virus [Watermelon mosaic virus [Zucchini tigré mosaic virus [Zucchini yellow mosaic virus [Plum pox virus-TAT (PPV-TAT) isolate, recombination breakpoints have been identified at two position in the entire CP gene encompassing the region coding for hypervariable N-terminal domain [Recombination and mutations are the two major factor in virus genome variability that is the driving force of virus evolution and variability of the viruses ,37. The ic virus  and Narc viruses , but intic virus , Canna yak virus , Lettuceic virus , Pea seeic virus , Plum poox virus ,53,54,55 virus Y ,57, Turnic virus , Watermeic virus , Zucchinic virus , Zucchinic virus ,61. Recol domain . Recombil domain . In the l domain ,47, but l domain . Our results extend the reported recombinant events within the potyvirus group describing for the first time intraspecies recombination in the case of tulip infecting potyviruses.Tulip mosaic virus  and TBV both are present as separate species in the ICTV database, GenBank, and Master Species List 2019.v1, but the two isolates (acc. # X63630 and KF442403) have high nt sequence identity (98.78%). Moreover, X63630 has 96.96% nt identity with TBV isolate BC24 (acc. #: MH886517)  and Tulip breaking virus-lily strain (acc. #: S44147) branch with LMoV strains, which is in agreement with previous reports [Based on the phylogenetic tree, aa and nt identity and differences in polyprotein cleavage sites, definitely three potyvirus species were identified on tulip plants in accordance with the ICTV species demarcation criteria. The latest species demarcation criteria in the case of potyviruses require nt identity less than 76% for the complete CP region to qualify as a different species . The exe reports ,22, so tLilium Asiatic hybrid analyzed in Japan (acc. #: AB674535), so Lily virus A should be a synonym of ReTBV.Based on the nt and aa sequence identities and phylogenetic analysis presented in this study, ReTBV should be elevated to species level (assigned as distinct species). Lily virus A (acc. #: JN127335) has 89.38 to 99.88% nt, and at least 95.49% aa identity in the CP region, with the ReTBV isolates described in this study , and also with the previously identified ReTBV from tulip derived from the Netherlands (acc. #: MG637051) and from Lilium species. In subgroup II all of the isolates originated from Lilium species. The isolates of ReTBV identified from tulips and lilies each also form a separate clade similarly to the LMoV strains. Due to the high bootstrap value, we propose to emerge subgroup I and II in ReTBV. Since limited data is available. it is in question whether real biological separation is in progress, so in the future, characterization of increasing number of isolates may answer this question. Previous analysis of a worldwide collection of Turnip mosaic virus (genus Potyvirus) demonstrated four phylogenetic lineages, mostly congruent with host types [The purpose of a phylogeny is to describe the evolutionary history of biological entities. According to the phylogenetic analysis of the tulip infecting potyviruses, the TBV isolates clearly form a distinct clade while LMoV and ReTBV strains formed a monophyletic group supported by high bootstrap values (100%). Within this monophyletic group the ReTBV and LMoV strains formed their own monophyletic group and they share a common ancestor. Our results support that LMoV isolates can be divided into two subgroups. Interestingly all of the strains isolated from tulip in Hungary and Japan grouped to subgroup I and in this subgroup only three isolates originated from st types ,63, so tThe extended analysis of potyviruses infecting monocotyledonous plants is primary interest, since monocotyledonous bulbs and grasses from Western Eurasia were assumed to be the primary and ancestral host for all potyviruses ,64,65. FSamples were collected during the flowering period of tulip plants showing tulip breaking syndrome from home and public gardens in different regions of Hungary during the growing seasons of 2017 and 2018. Permission to collect samples were asked in every case from the owners or head gardeners. For spatial distribution of viruses the samples were collected primarily from cultivar Apeldoorn (Darwin-hybrid group) ,66, the One gram of symptomatic leaf tissue was collected from each variety. Collected samples were transported on ice. Storage was carried out in a deep freezer at −70 °C ± 2 °C. Chenopodium amaranticolor seedlings grown in a vector free greenhouse. The final dilution of both the detection antibody and the enzyme conjugate were 1:100 (v:v). Plates were read at 405 nm wavelength on a Labsystems Multiskan MS ELISA reader after 15, 30, and 60 min incubation. A sample was considered positive if the absorbance of the sample was at least three times greater than that of the negative control.Potyvirus specific ELISA were carried out from the samples: antigen coated plate ELISA based on monoclonal antibody MAb PTY1   were car2 (5′-CGGGGATCCTCGAGAAGCTTTTTTTTTTTTTTTTT-3′). PCR reaction for 50 µL contained the following ingredients: 2 µL cDNA, 5 μL 10× Taq Buffer with KCl , 3 μL 25 mM MgCl2, 2 μL dNTPs, 10 mM each, 1-1 μL 100 µM poty7941 forward primer (5′-GGAATTCCCGCGGNAAYAAYAGYGGNCARCC-3′) and poly T2 as described earlier [Taq DNA Polymerase (recombinant) , and 35 μL nuclease free H2O. The PCR program for potyvirus detection started with initial denaturation for 3 min at 94 °C. The cycle continued with denaturation for 30 s at 94 °C, annealing primers for 30 s at 50 °C, polymerization at 72 °C for 2 min. The cycle was repeated 40 times, and a final polymerization step was included, 10 min at 72 °C. PCR products were visualized on 1% TBE agarose gels. Potato virus Y (acc. #: M95491) [®-T Easy vector  according to the manufacturer’s instruction. Plasmids were recovered with GeneJET Plasmid Miniprep Kit  and the sequence determination of the clones were carried out by service providers Biomi  and Base Clear BV. . To analyze whether a single potyvirus is present in the tulip plants, all of the original PCR products were digested with specific restriction endonucleases: in the case of TBV: NheI, ReTBV: HindIII, LMoV: SacII.Total RNA extraction was carried out according to the protocol of White and Kaper . Total R earlier , 1 μL Ta M95491)  maintain2) were cut off, assembled sequences were re-checked with megaBLAST to confirm identity.Each read was compared to sequences in the National Center for Biotechnology Information, U.S. National Library of Medicine (NCBI) GenBank using megaBLAST module  to identTobacco etch virus (RefSeq: NC_001555) was used as outgroup.For phylogenetic analysis alignment was built from the nucleotide (nt) sequences of acc. #: AB053256, AB078007, AB090385, AB674535, AF531458, AJ310203, EU267778, FJ618539, JN127335, JN127341, KF442403, KF553658, KJ561805, KT923168, MG637051, MF781080, MH360239, NC_001555, S44147, and X63630 using MEGA X  alignmenSequence identities were calculated in the alignment of CP sequences in MEGA X, using the p-distance matrix output.Tulip mosaic virus and Tulip breaking virus we used the following GenBank accession numbers for nucleotide alignment: KF442402, KF442403, KF826466, KT923168, MF983710, MH886517, MK368783, MK368785, MK368786, MK368787, MK368789, MK368791, MK368795, MK368796, MK368797, MK368799, MK368800, MK368805, MK368807, MK368808, X63630. The alignment was generated in CLC Genomics Workbench v20.0 (Qiagen) with less accurate option.In the case of For further evidence of taxonomic relations, the amino acid sequences at the NIb/CP cleavage sites were compared, where available. According to Adams et al. , we provp-value permutation number was 447, PhylPro [For recombination prediction the RDP v4.98 program ,83 was u PhylPro  was inst PhylPro  was run  PhylPro  and movi PhylPro  with Jin PhylPro  also use PhylPro , random  PhylPro  used int


Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production worldwide. Usually, it is detected in outbreaks of respiratory disease, most often during the winter period. During the middle of October 2018, a serious outbreak of respiratory disease occurred in a cattle farm comprising about 300 heads located in Central Italy. The herd was affected by a severe flu‐like syndrome unresponsive to any antibiotic treatment. Within 3 weeks, 39 adult animals died, and 12 abortions occurred. Direct and indirect laboratory tests were performed to detect the main pathogens causing the respiratory disease of the affected cattle. The results of laboratory investigations provided evidence of an acute and severe BRSV syndrome characterized by unusual mortality. In order to investigate the molecular underpinnings of this syndrome, phylogenetic analysis of the BRSV strain detected from the outbreak was carried out. The sequence analysis showed that the strain was genetically divergent from BRSV strains previously identified in Italy, as it showed high sequence similarity of more than 97% with strains isolated during a major BRSV epizootic that occurred in Sweden, Norway and Denmark during 2010–2011. The infection of the herd in Italy with this BRSV strain was likely due to the introduction of animals imported into Italy from abroad. Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production. In Italy, although respiratory problems due to BRSV are frequently described, major outbreaks have not yet been reported. Here, we report an outbreak caused by a BRSV strain with high mortality that occurred in a dairy farm in Central Italy; this strain shows a sequence similarity of >97% to the strains isolated during a major BRSV epizootic that occurred in Sweden and Norway during 2010–2011. Epidemiological analysis indicated that recently introduced heifers from Germany were the likely carriers of the virus. Globally circulating BRSV has a significant negative impact on the cattle industry due to economic losses because of the associated morbidity, mortality, costs of treatment and prevention, loss of production and reduced carcass value  belongs to the genus Bovine respiratory syncytial virus is an enveloped virus, and its genome contains 10 mRNAs, which are transcribed and translated into 11 viral proteins , bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea virus (BVDV), infectious bovine rhinotracheitis (IBR), and major bacterial agents that could have caused respiratory pathologies in cattle namely Mycoplasma spp. were carried out by conventional PCR ; Svanovir® BCV Ab (Boeringer Ingelheim Svanova); Prima Check® BRSV (Agrolabo Spa); Ab IDEXX® BVDV p80 Ab and IDEXX® IBRgB X3 (IDEXX Laboratories) following the manufacturer's instructions.Virological investigations were performed by conventional and real‐time PCR/RT‐PCR  and used as a template for the amplification of a 541‐bp region encoding the G protein. Amplification was performed by using a one‐step RT‐PCR kit (Qiagen) following the manufacturer's instructions, applying a previously published nested protocol (Vilcek, Elvander, Ballagi‐Pordány, & Belák, 3Mycoplasma spp., and two lungs for Mannheimia haemolytica. Serological investigations revealed the presence of antibodies against BPIV‐3 in four out of 20 animals, and against BRSV in all 20 animals. Based on the clinical and laboratory findings, the diagnosis was strongly in favour of acute BRSV syndrome. Regarding abortions, infectious agents associated with abortion in cattle were not investigated. On the other hand, the abortifacient role of BRSV has never been demonstrated, so we assume that the respiratory illness might have caused abortion secondary to the systemic infection, as it can occur for any disease which results in a sick cow and a high fever, although that can only be speculated.Overall, the pathological examination showed tracheal congestion, fibro‐purulent bronchopneumonia, marked oedema and interstitial emphysema characterized by consolidation of the apical and middle lobes of the lungs. The results of laboratory investigation are presented in Table The sequence analysis conducted on two PCR‐positive BRSV samples Figure , showed Mycoplasma spp. or Mannheimia haemolytica.We reported an outbreak with high mortality that occurred in a dairy farm in Central Italy and was attributed to a BRSV strain never described in the country. This strain had extremely negative effects on herd health and animal performance, as it had induced a rather high mortality and severe signs of respiratory disease. In few samples from lungs BRSV was detected together with either After the diagnosis, an emergency vaccination plan was set up with a live attenuated BRSV vaccine, which was administered intra‐nasally. Nevertheless, the animals continued to die. Live and inactivated vaccines against BRSV are generally used, but their efficiency has been questioned (Valarcher et al., The BRSV strain characterized in this study did not cluster together with the viruses previously identified in Italy, but it belonged to subgroup II with isolates collected in Sweden, Norway and Denmark during a major BRSV epizootic that occurred during 2010–2011. As a result, in Italy, at least three distinct groups of BRSV have been detected so far, with our strain representing a group with particularly high virulence. The recent introduction to the herd of 22 heifers that came from Germany, which has frequent commercial exchanges with northern European countries, likely explains the circulation of this new BRSV strain on the farm. Viruses from areas where vaccination is heavily used show frequent amino acid changes, which may lead to the emergence of strains that can potentially evade the immunity conferred by the vaccine. Our study also raises an important question about the adequacy of the available diagnostic tools and implications for control programs. Continuous investigation and molecular characterization of positive samples collected in the country are useful tools for updating knowledge on BRSV epidemiology and evolution of the virus during the time. Moreover, we could notice the emergence of new viral variants that may escape vaccination protection. The successful control and eradication of a virus is possible when the epidemiology of the virus infection is clear and accurate diagnostic methods are available. In addition, an update of the vaccines is also recommended.The authors declare no conflicts of interest.Monica Giammarioli: conceptualization; data curation; formal analysis; writing‐original draft. Piermario Mangili and Alex Nanni: investigation. Ilaria Pierini, Silvia Pirani and Paola Gobbi: methodology; software; validation. Stefano Petrini: methodology; supervision; validation. Gian Mario De Mia: supervision; writing‐review & editing.The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethics review committee approval has been received.


When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance. a priori knowledge and the utilization of appropriate assays for their detection. Mosquitoes can also provide other valuable information, including unexpected or novel arboviruses, nonarboviral pathogens ingested from hosts they feed on, and their own genetic material. Metagenomic analysis using next-generation sequencing (NGS) is a rapidly advancing technology that allows us to potentially obtain all this information from a mosquito sample without any prior knowledge of virus, host, or vector. Moreover, it has been recently demonstrated that pathogens, including arboviruses and parasites, can be detected in mosquito excreta by molecular methods. In this study, we investigated whether RNA viruses could be detected in mosquito excreta by NGS. Excreta samples were collected from Aedes vigilax and Culex annulirostris experimentally exposed to either Ross River or West Nile viruses and from field mosquitoes collected across Queensland, Australia. Total RNA was extracted from the excreta samples, reverse transcribed to cDNA, and sequenced using the Illumina NextSeq 500 platform. Bioinformatic analyses from the generated reads demonstrate that mosquito excreta provide sufficient RNA for NGS, allowing the assembly of near-full-length viral genomes. We detected Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and identified seven potentially novel viruses closely related to members of the order Picornavirales (2/7) and to previously described, but unclassified, RNA viruses (5/7). Our results suggest that metagenomic analysis of mosquito excreta has great potential for virus discovery and for unbiased arbovirus surveillance in the near future.Traditional screening for arboviruses in mosquitoes requires IMPORTANCE When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance. Wolbachia, from a single mosquito in a single reaction  that feed on the blood of a variety of vertebrate hosts . Mosquitreaction . Viral mreaction , and mesreaction  from fieWuchereria bancrofti after mass drug administration programs , Ross River virus (RRV), and West Nile virus (WNV) have been detected in mosquito excreta by reverse-transcription real-time PCR (RT-rtPCR) by us and others , 14. AddIn this study, we evaluated the application of NGS for unbiased detection of RNA viruses, first in excreta harvested from experimentally infected mosquitoes and, later, in excreta of field-collected mosquitoes sampled from different locations in the state of Queensland, Australia. The sampling locations in Queensland have a history of transmission of medically important arboviruses, such as RRV, WNV, and Barmah Forest virus , 18, as Culex annulirostris or Aedes vigilax mosquitoes exposed to either WNV (Kunjin subtype [WNVKUN]) or RRV, by swabbing a Parafilm disc placed in the bottom of each container  cycle threshold (TC) values of 26.3 ± 0.4 and 26.8 ± 1.0, respectively. Based on these results, six libraries were subsequently prepared and next-generation sequenced using the Illumina NextSeq platform  of mosquitoes experimentally infected with arboviruses provide sufficient template for NGS, allowing the assembly of near-full-length viral genomes , which coincidentally was also positive for RRV. A total of 703 female mosquitoes from the two traps that contained the two RRV- and/or BFV-positive excreta samples mentioned above were sorted into 42 pools of ≤50 mosquitoes and screened for RRV and BFV using RT-rtPCR. Five (12%) pools were positive for RRV, and five (12%) pools were positive for BFV  and Cattana Wetlands (FNQ21), far North Queensland, Australia, processed for virus detection from traps that yielded excreta samples positive for Ross River and Barmah Forest viruses. Download Copyright © 2020 Ramírez et al.2020Ramírez et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00587-20.4FIG S1FIG S1, EPS file, 1.0 MB.Study sites in Queensland, Australia, where mosquitoes and mosquito excreta were collected. (A) Far North Queensland (FNQ); (B) South East Queensland (SEQ). Download Copyright © 2020 Ramírez et al.2020Ramírez et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00587-20.5DATA SET S1Data Set S1, XLSX file, 0.02 MB.Mosquito species identified in this study. Download Copyright © 2020 Ramírez et al.2020Ramírez et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A total of 47 libraries , corresponding to excreta samples collected from 11 locations, were sequenced using the Illumina NextSeq platform, with a mean (± SEM) of 12,776,515 ± 565,467 raw reads per sample . Upon prPicornavirales and previously described unclassified RNA viruses. For three of the viruses detected  only partial sequences were obtained; for the rest of the viruses, we were able to assemble near-full-length genomes. Of these, Australian Anopheles totivirus (AATV), Wuhan insect virus 33 (WIV33), and Hubei odonate virus 5 (HBOV5) showed >90% amino acid (aa) identity over the RNA-dependent RNA polymerase (RdRp) to published sequences in GenBank, indicating that they correspond to strains of these viruses Fig. 3). Culex mosquitoes from Korea and California, respectively  sequences related to both Culex Daeseongdong-like virus and Daeseongdong virus 2 (DV2) . The seqectively , 27. OurThe FNQ collections appeared to have greater virus diversity, with full-length sequences from 6 different viruses identified only from FNQ , compared with 4 identified only from SEQ . Only OPTV was detected in both FNQ and SEQ samples.n = 1,000) against SILVA small-subunit (SSU) and large-subunit (LSU) rRNA sequences databases  and were likely subjected to less sample degradation than field samples. Since the viral titers to which mosquitoes were exposed to during laboratory infection may have been higher than those encountered by mosquitoes in the wild, future investigations should include determination of the limit of detection for this method by exposing mosquitoes to different viral titers.Over the past decade, unbiased metagenomic analysis using NGS has become a valuable tool and revolutionized virus discovery and surveillance , 30. HowTC values of ≤28.5. Among the libraries that were positive by RT-rtPCR for RRV and/or BFV but negative by NGS, all had TC values of ≥36.0, suggesting that the failure to detect these viruses may have resulted from low abundance of viral sequences. Further, our results indicate a large amount of rRNA present in mosquito excreta, with an average of more than 50% of sequence reads matching both SSU and LSU rRNA, which could potentially impact the sensitivity of virus detection. In the future, it is likely that by adding an rRNA depletion step, sensitivity can be increased. Additionally, viral enrichment protocols such as NetoVIR  had been previously identified in mosquitoes and four  were related to viruses previously identified in mosquitoes. Some of these viruses have been isolated and/or characterized. In the case of AATV, although the virus has not been isolated in C6/36 mosquito cells, evidence of its replication in mosquitoes has been observed by small-RNA analysis . ArIFV, n silico , 26, 27.Although these insect-borne viruses are unlikely to be associated with disease in vertebrates, they can potentially affect the vector competence of mosquitoes for pathogenic viruses, as demonstrated previously for a number of insect-specific viruses , 39. BecAs of 2020, the costs associated with NGS and the time and bioinformatics skills required to analyze the results from thousands to tens of thousands of mosquitoes can be prohibitive, especially in low-resource settings . We havein vitro. Use of traps that are highly selective for mosquitoes, such as CO2-baited passive box traps that do not deploy lights that attract other insects, is recommended  containing either WNV  or RRV (108.1±0.1 TCID50/ml) via the hanging drop method  for 3 min at 26 Hz and centrifuged at 14,000 × KUN . Every run included synthetic primer and probe controls, a positive extraction control , a negative extraction control, and a no-template control (molecular-grade water). The results were evaluated qualitatively: for any sample, a threshold cycle number (TC) of >40 indicated that no RNA was detected . The newly synthesized DNA was purified by ethanol precipitation. DNA libraries were prepared using the Nextera XT DNA library preparation kit (Illumina) and Illumina Nextera XT index kit. The resulting libraries were analyzed, and DNA sizing and quantification were performed using a 2200 TapeStation (Agilent Technologies). A fetal calf serum (FCS) library was prepared as described above from FCS RNA as a negative control. Libraries were diluted to 1 nM, pooled, denatured and diluted to a final concentration of 1.2 pM. Paired-end sequencing was performed using the NextSeq platform (Illumina) using a NextSeq 500 Mid Output V2 kit (Illumina) (Genomic DNA (gDNA) was removed from total RNA aliquots extracted without carrier RNA using the Heat & Run gDNA removal kit . RNA was reverse transcribed, and first-strand cDNA was synthesized using the NEB Protoscript II first-strand cDNA synthesis kit  according to the manufacturer’s instructions, followed by second-strand cDNA synthesis using NEB second-strand synthesis enzyme buffer  and second-strand DNA enzymes (DNA polymerase I [llumina) .http://sapac.support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html). An initial search of the raw sequences was conducted using DIAMOND (BLASTx)  and short (<100-nt) reads were trimmed using BBDuk before being assembled using SPAdes (Sequence reads were demultiplexed and adapters were removed using bcl2fastq version 2.20 ((BLASTx)  against (BLASTx)  using thg SPAdes  with defg SPAdes  and usedg SPAdes . This prAssembled sequences with > 90% amino acid identity with existing viruses over the RNA-dependent RNA polymerase (RdRp) were considered strains of these viruses and assumed to phylogenetically group with them. Because of this, only complete sequences with less than 90% amino acid identity to the reference sequence were included in phylogenetic analyses. The translated contigs were aligned with protein sequences obtained from GenBank using the results from BLAST and previously published phylogenetic trees of the related viruses , 25. Muln = 1,000) from all the excreta samples shown to contain a virus by NGS (from both laboratory-infected and field-collected mosquitoes) by performing a BLAST search (https://www.arb-silva.de/)   using GePRJNA631724. All virus genome sequences generated in this study have been deposited in GenBank under accession numbers MN784056 to MN784081.Raw sequence reads generated from this study have been deposited in the NCBI Sequence Read Archive (SRA) repository under project number


Riptortus pedestris, infected with the entomopathogenic fungus Beaveria bassiana JEF-007. The complete genome sequence was 9,915 nucleotides long and encoded a 2,916-amino-acid polyprotein. This virus belonged to Iflaviridae based on phylogenetic analysis and was named RiPV-2.A novel putative single-stranded RNA virus was discovered from the transcriptome of a bean bug,  Riptortus pedestris, infected with the entomopathogenic fungus Beaveria bassiana JEF-007. The complete genome sequence was 9,915 nucleotides long and encoded a 2,916-amino-acid polyprotein. This virus belonged to Iflaviridae based on phylogenetic analysis and was named RiPV-2.A novel putative single-stranded RNA virus was discovered from the transcriptome of a bean bug,  Riptortus pedestris (Hemiptera: Alydidae) is a serious soybean pest in the Republic of Korea and Japan  were filtered using FastQC, and adaptors were trimmed using Trimmomatic  to known viruses. One of the viral sequences was previously characterized and named RiPV-1 in 2016  insect virus of bean bugs named RiPV-2.The second assembled viral sequence  was subjected to an NCBI blastx search, and the first hits were Hubei picorna-like virus 33, which is an unclassified RNA virus with 46.2% identity  with a coverage of 371× and a GC content of 36%. Both the 5′ and 3′ ends were confirmed using the rapid amplification of cDNA ends (RACE) technique , 9. The Brevicoryne brassicae virus-UK, and Darwin bee virus 3, with identities of 64%, 45%, 44%, 41%, and 40%, respectively. The phylogenetic analysis based on the RdRp motif and helicase motif revealed that RiPV-2 represents a distinct species within the genus Iflavirus, closely related to Bovine rhinitis B virus (BrBV), Perina nuda virus (PnV), and Ectropis obliqua picorna-like virus (EoV) (Marnaviridae (The RdRp amino acid sequence of RiPV-2 showed the highest-similarity score (95∼99% query coverage) with Hubei picorna-like virus 33 , Shahe hus (EoV) , while taviridae .R. pedestris, and belongs to unclassified Picornavirales.In conclusion, the sequence of RiPV-2 represents a novel RNA virus with distinct genomic characteristics compared to RiPV-1, which was found in the same insect host, MN078225. The raw data have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRP068540.The complete genome sequence of the RiPV-2 genomic RNA has been deposited in GenBank under the accession number


Alzheimer’s disease and related dementias (ADRD) affects 5.5 million Americans, and is expensive despite the lack of a cure or even treatments effective in managing the disease. The literature thus far has tended to focus on the costs to Medicare, despite the fact that one of the main characteristics of ADRD (the loss of independence and ability to care for oneself) incurs costs not covered by Medicare. In this paper, we use survey data for 2002-2014 from the Health and Retirement Study to estimate the out-of-pocket costs of ADRD for the patient and their family through the first 8 years after onset of symptoms, as defined by a standardized 27-point scale of cognitive ability. A two-part model developed by Basu and Manning (2010) allows us to separate the costs attributable to ADRD into two components, one driven by differences in longevity and one driven by differences in utilization. We consider total out-of-pocket expenditures, as well as out-of-pocket expenditures by category . Our results suggest that the out-of-pocket costs of ADRD are quite substantial over the first 8 years after onset. We also find that out-of-pocket spending is decreasing over the first 8 years, similar to the trend seen in Medicare expenditures. The results of this study highlight the financial burden of ADRD, particularly for the population paying out-of-pocket for care.


Cervical disc arthroplasty (CDA) has been demonstrated, in clinical trials, as an effective and safe treatment for patients diagnosed with radiculopathy and/or myelopathy. However, the current CDA indication criteria, based on the preoperative segmental range of motion (ROM), comprises a wide range of variability. Although the arthroplasty level preserved ROM averages 7°-9° after CDA, there are no clear guidelines on preoperatively limited or excessive ROM at the index level, which could be considered as suitable for CDA.This was a retrospective study of patients who underwent CDA between January 2008 and October 2018 using Prestige-LP discs in our hospital. They were divided into the small-ROM (≤5.5°) and the large-ROM (> 12.5°) groups according to preoperatively index-level ROM. Clinical outcomes, including the Japanese Orthopedics Association (JOA), Neck Disability Index (NDI), and Visual Analogue Scale (VAS) scores, were evaluated. Radiological parameters, including cervical lordosis, disc angle (DA), global and segmental ROM, disc height (DH), and complications were measured.P = 0.046). Patients in both groups had significantly improved JOA, NDI, and VAS scores after surgery, but the intergroup difference was not significant. Patients in the small-ROM group had dramatic postoperative increase in cervical lordosis, global and segmental ROM (P < 0.001). However, there was a paradoxical postoperative decrease in global and segmental ROM in the large-ROM group postoperatively (P < 0.001). Patients in the small-ROM group had lower preoperative DH (P = 0.012), and a higher rate of postoperative heterotopic ossification (HO) (P = 0.037).One hundred and twenty six patients, with a total of 132 arthroplasty segments were analyzed. There were 64 patients in the small-ROM and 62 in the large-ROM group. There were more patients diagnosed with cervical spondylosis in the small-ROM than in the large-ROM group (Patients with preoperatively limited segmental ROM had severe HO, and achieved similar postoperative clinical outcomes as patients with preoperatively excessive segmental ROM. Patients with preoperatively limited segmental ROM showed a postoperative increase in segmental mobility, which decreased in patients with preoperatively excessive segmental ROM. In recent decades, cervical disc arthroplasty (CDA) has been studied in many clinical trials as an alternative surgical treatment to anterior cervical discectomy and fusion (ACDF), due to a paradigm shift towards preserving motion and avoiding adjacent segment disease –7. SegmeThere is limited data to answer the question above. Tu et al.  concludePatients (312) who underwent CDA or hybrid surgery (HS) in the West China Hospital of Sichuan University, using Prestige-LP discs between January 2008 and October 2018, were retrospectively reviewed. The surgical indications were intractable symptomatic radiculopathy and/or myelopathy caused by cervical degenerative disc disease (DDD) or spondylosis at 1-or 2-levels, from C3-C7. Exclusion criteria for arthroplasty were: 1) severe facet joint degeneration, 2) ossification of the posterior longitudinal ligament (OPLL), 3) segmental ROM < 2°, 4) segmental instability , 5) intervertebral disc height loss more than 50% and, 6) less than 12 months follow-up period. ACDF was performed if radiographic signs of instability, bridging osteophytes, and severe facet degeneration were observed in the 2-level disease. The study protocol was approved by the institutional ethics committee, and all patients signed informed consent.P = 0.200). Based on the raw data, the cutoff values for preoperative small ROM were defined as mean - SD, while large ROM was mean + SD. Therefore, the small-ROM group was defined as having segmental ROM of ≤5.5° at the index level . A standard right-sided anterior cervical approach was performed after general anesthesia. Thorough decompression was done at the index levels by removing the disc tissue, posterior longitudinal ligament, and osteophytes to achieve neural decompression. Appropriated Prestige-LP disc  or Zero-P implant  was inserted into the intervertebral space after the endplates were well prepared. Then, C-arm fluoroscopy was performed to verify the proper placement of the implants. A drainage tube was inserted after irrigation and hemostasis before the closure of the incision.The clinical and radiographic outcomes of patients were routinely evaluated at regular intervals: before surgery, at 1 week, 3, 6, 12 months postoperatively, and at the last follow-up. Clinical outcomes were evaluated according to validated self-assessment questionnaires, including the Japanese Orthopedics Association (JOA), Visual Analogue Scale (VAS), and Neck Disability Index (NDI) scores. Radiological parameters including cervical lordosis, disc angle (DA) of the arthroplasty segments, ROM of C2 - C7 and the arthroplasty segments, and disc height (DH) were measured in lateral radiographs in neutral, extension and flexion views. Global and segmental ROMs were defined as the difference in respective Cobb angles between flexion and extension views. We applied the McAfee classification system (Grades 0 to 4) to classify heterotopic ossification (HO) . McAfee p < 0.05.Statistical analysis was performed using SPSS software version 19.0 . The results were presented as mean  ±  SD or percentages. A paired t-test was used to compare preoperative and postoperative parameters. The independent t-test or the Mann–Whitney U test was used to compare continuous variables between the two groups. The Chi-square or Fisher’s exact test was used for inter-group categorical variables. One-way ANOVA was used for continuous variables among the three surgical types. Tests were two-tailed with n = 75), followed by C4/5 (n = 36), and then C6/7 (n = 21). No patients underwent 2-level CDA distributed target levels in the different groups.One hundred and 26 patients underwent 1-or 2-level surgery for a total of 132 arthroplasty segments (Table P < 0.001) in both the small-ROM and large-ROM groups. However, there were no significant differences in the JOA, NDI, and VAS scores between the two groups at any follow-up point  for the small-ROM group, but changed slightly in the large-ROM group (12.01° ± 12.62° vs.12.06° ± 9.08°), respectively. Patients in the large-ROM group showed significantly larger cervical lordosis before surgery (P = 0.008), but there was no significant post-surgical difference between the two groups. The overall cervical motion increased significantly from admission to the last follow-up in the small-ROM group  and significantly decreased in the large-ROM group . The changes over the follow-up period are shown in Fig. The preoperative cervical lordosis values (6.25° ± 11.24°) had increased significantly at the last follow-up , and tended to have less reduction  at the last follow-up.The small-ROM group showed significantly less preoperative disc lordosis than the large-ROM group . The change of segmental ROM at the arthroplasty level was similar for different surgical techniques (Table There was a significant increase in the preoperative (4.05° ± 1.04° to 7.11° ± 3.43°) for the small-ROM group at the last follow-up, for an overall delta ROM (△ROM) of 3.05° ± 3.69° (P = 0.012).The average pre- and postoperative DH in the small-ROM group were 5.22 mm ± 0.81 mm and 6.40 mm ± 0.70 mm, respectively. The corresponding values in the large-ROM were 5.59 mm ± 0.85 mm and 6.60 mm ± 0.77 mm, respectively. There was a significant difference in DH between the two groups preoperatively (p = 0.037). Although the proportion of high-grade HO levels was higher in the small-ROM as compared to the large-ROM group, the difference was not significant . We divided the arthroplasty levels into positive △ROM (A) and a negative △ROM subgroups (B). Fourteen levels with less mobile (≤5°) were in subgroup B; however, 11 of them (78.6%) developed high-grade HO  in the small-ROM and 10 (16.1%) in the large-ROM group had degenerative radiographic changes Table . The ratMany trials have substantiated the theoretical advantages of CDA over fusion in recent decades, such as preservation of motion through the segments that were operated on; however, the documented ROM of CDA shows wide variability among patients. Several investigations suggest that the preoperative ROM scale is attributed to the variability of ROM after CDA , but theThere was a difference in radiographic features between the small and large-ROM groups. Patients with limited segmental ROM showed significantly less global and segmental lordosis, ROM, and shorter DH. This may be due to the degenerative cascade concept; that loss of proteoglycans and water in the nucleus pulposus causes disc height loss, leading to excessive motion and instability at the early stage of disc degeneration and loss of segmental ROM at the late stage. These patients also suffered from relatively severe cervical spine degeneration. In the current study, we found that there was no significant difference in postoperative segmental ROM at the arthroplasty level among the three surgical types. The trends of segmental ROM did not alter regardless of the arthroplasty level adjacent to a fusion mass or a artificial disc. This result indicates that the change of ROM at each index level is relatively independent after surgery.Disc with preoperatively limited ROM showed a significant increase in △ROM, by 3.05°, which parallels the observations of a previous study by Tu et al. ; howeverThe key concern for patients who had excessive preoperative ROM was that the associated hypermobility would cause increased stress loading on the facet joints and accelerate their degeneration, leading to additional neck pain. However, based on the clinical and radiographic outcomes, we observed that segmental mobility preservation at the index level and the maintenance of motion through the posterior elements did not place patients at risk of increased neck pain. Thus, we proposed that selected patients with preoperatively limited or excessive segmental ROM were good candidates for CDA.Although there is no consensus on the mechanism of HO, its development has been associated with variables such as age, sex, disc height, residual exposed endplate, and mismatch of the prosthesis , 23, 24.There were several limitations to the study. First, it was retrospective and carried out at a single institution, presenting inherent weaknesses and limited generalizability of the findings. Second, we evaluated the disc-levels as long as they met the inclusion criteria before surgery. However, different surgery types or disc-levels in the subaxial cervical spine may affect outcomes. The small sample size did not present adequate-subgroup data to cover all potential factors. In the current study, factors such as age, sex, and primary cause did not have any significant effect on the results, other than a tendency between the two groups, which may also attribute to the small sample size. Third, the study was limited to the Prestige-LP discs range of motion, whose FDA defined inclusion criterion of segmental ROM is 2°-20°; thus, the results may not represent any other type of prostheses. Fourth, HO formation was a time-dependent complication after CDA. The results may not have been precisely evaluated due to the extensive period of the study and the relatively small sample of long-term follow-up cases.Patients with preoperative limited segmental ROM had significantly severe postoperative HO and similar clinical improvents as patients with preoperative excessive segmental ROM. However, patients with preoperative limited segmental ROM showed increased postoperative segmental mobility, whereas patients with preoperative excessive segmental ROM paradoxically exhibited decreased postoperative segmental mobility. With proper patients selection, discs with limited or excessive mobility could benefit from CDA.


Arabidopsis by Pseudomonas sp. strains associated with rhizosphere of crop plants grown in Mekong Delta, Vietnam. Out of all the screened isolates, Pseudomonas PS01 isolated from maize rhizosphere showed the most prominent plant growth promoting effects on Arabidopsis and maize (Zea mays). We also found that PS01 altered root system architecture (RSA). The full genome of PS01 was resolved using high-throughput sequencing. Phylogenetic analysis identified PS01 as a member of the Pseudomonas putida subclade, which is closely related to Pseudomonas taiwanensis. PS01 genome size is 5.3 Mb, assembled in 71 scaffolds comprising of 4820 putative coding sequence. PS01 encodes genes for the indole-3-acetic acid (IAA), acetoin and 2,3-butanediol biosynthesis pathways. PS01 promoted the growth of Arabidopsis and altered the root system architecture by inhibiting primary root elongation and promoting lateral root and root hair formation. By employing gene expression analysis, genetic screening and pharmacological approaches, we suggested that the plant-growth promoting effects of PS01 and the alteration of RSA might be independent of bacterial auxin and could be caused by a combination of different diffusible compounds and volatile organic compounds (VOCs). Taken together, our results suggest that PS01 is a potential candidate to be used as bio-fertilizer agent for enhancing plant growth.The objectives of this study were to evaluate the plant growth promoting effects on  Pseudomonas is one of the most abundant genera of PGPR, which is capable of enhancing plant growth and modulating the root system architecture in different plant species  was checked on Pikovskaya’s agar , 0.5 mM K3[Fe(CN)6], 0.5 mM 5-bromo-4-chloro-3-indolyl-b-glucuronic acid, and 0.01% Silwet L-77) at 37 °C for 2 h  was wasAIR12nd NAC1) . We founAAO1, ASA1, TSA1, YUCCA5, CYP79 B2, CYP83 B1 and IGPS) [AAO1, ASA1, TSA1, YUCCA5, CYP79 B2, IGPS showed no changes after inoculation with PS01. The expression level of CYP83 B1 was slightly down-regulated in the roots after 24 h of co-cultivation while it was not significantly affected after 48 h of co-cultivation. Inoculation with PS01 down-regulated CYP83 B1 expression in shoot after 24 h of co-cultivation but induced an increase in its transcript abundance after 48 h of co-cultivation   were anativation . TherefoArabidopsis, including AUX1, IAA3/SHY2, PIN1, PIN2 [IAA3 which was slightly down-regulated in the shoot 48 h after inoculation  is the result of a polar auxin transport in plants. To evaluate the role of auxin transport on the alteration of RSA, an inhibitor of auxin polar transport, NPA was used. NPA inhibits root elongation and differential growth as well as lateral root development. Our results showed that the LRs formation was completely blocked in the presence of 1µM NPA. Furthermore, the PS01-induced LRs formation was affected in the presence of NPA. In PS01-inoculated seedlings, the number of LRs in the absence of NPA was 8.5 while that of NPA-treated seedlings was 6.5 a. In thePseudomonas strain PS01 was isolated from maize rhizosphere; therefore, we also test the effects of this bacterial strain on the seed germination and the growth of maize plants under nutrients deficient condition were analyzed. The results showed that after 2 days of incubation, a higher rate of germination was observed in bacteria coated seeds (51.5%), compared to uncoated seeds (31.3%) (n = 100)  compare to non-inoculated roots (9.7 × 101 g−1 of fresh roots) (The n = 100) . Previou by PS01 . Here, wh roots) . These rPseudomonas species are widely distributed in soil and plant roots. Indeed, Pseudomonas can be isolated successfully from almost every kind of agriculture soil or plant root. However, the inoculated bacteria sometimes cannot survive or perform in the same way in other soil and climatic conditions, because they must compete with the better-adapted indigenous microflora [Pseudomonas isolated and characterized in the Mekong Delta [Arabidopsis seedlings.croflora ,55,56. Tng Delta ,45. In tP. aeruginosa is an opportunistic human pathogen, whereas other species of Pseudomonas are non-pathogenic when used in agriculture [Pseudomonas putida subclade. Interestingly, P. putida is classified in the risk group-1/BSL-1 class, which has been reported to have relative low harmful effects on human and the environment [P. putida strains have been reported in humans, however, these are rare and mostly reported in immuno-compromised individuals [Prior to application of microorganisms for agronomic purposes as a bio-inoculant, scenario safety testing and risk assessment evaluation was required. For instance, iculture ,57. In tironment . In few ividuals . TherefoArabidopsis RSA by inhibiting primary root elongation and promoting lateral root and root hair formation. These results are in agreement with the findings of earlier studies on P. fluorescens WCS417, P. fluorescens WCS374, P. putida WCS358, S. marcescens and B. megaterium [Pseudomonas isolates [To understand and quantify the impact of PGPRs on roots and the whole plant, a common strategy was to inoculate roots with a PGPR In vitro and quantify effects on plant development. This methodology has shown that a large number of PGPRs may modify the RSA as well as root functionality, leading to the increase in uptake of nutrients and water and improve the overall growth of the whole plant . A commogaterium ,60. In aisolates .TIR1/AFB auxin receptor inhibitor (auxinole) and was not affected in the auxin mutants axr4-1, eir1-1, and aux1-7. The amount of auxin produced by PGPRs was directly correlated to the plant growth promotion and root development in a dose dependent manner. Interestingly, the alteration of root system architecture in response to P. fluorescens WCS417 and B. megaterium are similar to PS01 despite the fact that these PGPRs cannot produce IAA [P. brassicacearum STM196 strain was compared with the high-IAA-producing A. brasilense sp245 strain and its low-IAA-producing ipdC mutant. STM196 was found to not release significant amounts of auxin in the rhizosphere [Changes in RSA by PGPRs mainly resulted from their ability to interfere with the plants hormonal balance, implicating the production of phytohormones and other signals . IAA is duce IAA ,60. In azosphere .Pseudomonas spp. colonizing plant roots including P. chlororaphis; P. fluorescens and P. putida [A. brasilense can produce NO during root colonization [Pseudomonas WCS417 are not involved in WCS417-induced inhibition of primary root elongation but play an important role in promotion of LR formation [P. putida and P. fluorescens was reported [Some bacterial compounds other than auxin can also alter RSA in the same way as PS01 . The bac. putida . Many PGnization ,67,68,69nization . Indeed,nization . VOCs prormation . RecentlArabidopsis DR5::GUS reporter line. This result is in agreement with the findings of earlier studies on P. brassicacearum STM196 and Pseudomonas WCS417 [B. subtilis GB03 can promote the growth of Arabidopsis seedlings by regulating auxin homeostasis in GB03-exposed roots as revealed by the DR5::GUS auxin-responsive Arabidopsis line [Arabidopsis via VOCs, which could act in an auxin-dependent or auxin-independent pathway. More than one secreted metabolite from strain PS01 may be involved in alteration of RSA. Therefore, screening for candidate strains based on a particular plant growth promoting trait such as IAA production can lead to overlooking many promising strains.Although it remains unknown whether auxin is a direct or in direct signalling molecule in alteration of RSA, most research groups suggested that auxin transport and signaling in plants may play an important role. Both primary-root elongation and lateral-root formation require auxin transport and an auxin gradient in the primary root tip . A changs WCS417 ,43. The sis line . This stPseudomonas PS01 strain as phytostimulator was demonstrated. Pseudomonas PS01 improved seed germination and promoted vegetative growth in maize. These results reinforced the idea that PS01 could be used for the formulation of a new bio inoculant. Previously, we reported the ability of the PS01 strain in inducing a salt tolerance response in Arabidopsis thaliana [Pseudomonas when applied in practical agriculture are related to their ability to colonize the rhizosphere and persistence throughout the growing season. Because they can exist in the natural habitat of the rhizospheres, they could colonize root surface when reintroduced through inoculation. Furthermore, the ability of PS01 to colonize and survive in rhizosphere should be assessed. Currently, the whole genome of PS01 had been sequenced, providing maximum of phylogenetic information for precise identification. Therefore, investigation of safety assessments such as presence of pathogenicity or virulence related genes may be also determined. In the future, it will be critical to conduct a more safety studies of Pseudomonas PS01 before biotechnological applications of this strain can be envisaged. Collectively, our results suggest that Pseudomonas PS01 can be developed for field application as an alternative to chemical fertilizers. Prediction and annotation gene sequences have been carried out to provide an in-depth view of the PGP characteristics of this strain, highlighting its potential use as a bio inoculant in agriculture.In our study, the potential of the thaliana . However


There is concern that using depot-medroxyprogesterone acetate (DMPA) for pregnancy prevention heightens HIV susceptibility. While no clinical data establishes causal link between HIV acquisition and use of this injectable progestin, prior work from our laboratory showed that DMPA comparably lowers genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and weakens genital epithelial barrier function in female mice and women. We likewise saw DMPA increase mouse susceptibility to multiple genital pathogens including HIV. Herein, we sought to confirm and extend these findings by comparing genital epithelial barrier function in untreated rhesus macaques (RM) vs. RM treated with DMPA or DMPA and estrogen (E). Compared to controls, genital tissue from RM with pharmacologically relevant serum levels of medroxyprogesterone acetate displayed significantly lower DSG1 levels and greater permeability to low molecular mass molecules. Conversely, DMPA-mediated effects on genital epithelial integrity and function were obviated in RM administered DMPA and E. These data corroborate the diminished genital epithelial barrier function observed in women initiating DMPA and identify RM as a useful preclinical model for defining effects of exogenous sex steroids on genital pathogen susceptibility. As treatment with E averted DMPA-mediated loss of genital epithelial barrier function, our results also imply that contraceptives releasing progestin and E may be less likely to promote transmission of HIV and other sexually transmitted pathogens than progestin-only compounds. High rates of maternal and infant mortality and the need for affordable contraception are characteristics common to many parts of the world with large burden of HIV disease, and these conditions combine to make control of the HIV pandemic and pregnancy prevention highly interconnected public health concerns . EntwiniWhile adolescent and young women in SSA are disproportionally affected by HIV, the reasons for this are not well defined. However, various explanations have been offered, including gender-based social, economic, political, and cultural disparities, and the high regional prevalence of intergenerational sexual relationships . In addiAs rationale for the disproportionate impact of HIV on adolescent and young women in SSA remains ambiguous, additional clinical studies that minimize the impact of confounding behavioral variables on data interpretation are needed. It is also necessary to precisely define biological mechanisms with the potential to alter HIV transmission efficiency in young reproductive-age women. Our laboratory showed that treating mice with DMPA reduced genital levels of desmoglein-1 (DSG1) and other cell-cell adhesion molecules and increased penetration of intravaginally administered low molecular mass molecules and activated leukocytes into genital submucosal tissue . We alsoMacaca mulatta) used in these studies were 5–8 years old and were housed at the California National Primate Research Center. All studies were approved by the University of California–Davis Institutional Animal Use and Care Committee prior to initiation and each procedure performed in accordance with regulations established by the American Association for Accreditation of Laboratory Animal Care. For sedation, RM were intramuscularly (IM) administered 10 mg/kg of ketamine hydrochloride  or 0.7 mg/kg of tiletamine hydrochloride and zolazepam . After adequate sedation, 10 mL of peripheral blood was collected at indicated time points to quantify serum levels of progesterone (P4), estradiol (E2), and medroxyprogesterone acetate (MPA)  and stored at −80°C for use in gene expression studies. The second ectocervical biopsy was placed in 4% buffered methanol-free formaldehyde  and used in histological and immunohistochemical studies.Female RM (te (MPA) . Two vagAs indicated in 4 and E2 levels were measured by electrochemiluminescence [The Endocrine Technologies Support Core at the Oregon National Primate Research Center quantified serum sex steroid concentrations. Using a Cobas® e 411 Analyzer , Pnescence , 16. MPAnescence .2. Specimens were fixed 24 h in buffered 4% methanol-free formaldehyde and embedded in 6% agarose. Using a PELCO EasiSlicer™ , 200–300 μm tissue sections were counterstained with DAPI . Sections were viewed with a Nikon A1 confocal microscope and vaginal mucosal epithelial permeability calculated by fluorescent LY molecule infiltration of the vaginal epithelium (pixels/μm2) using ImageJ software [Evaluation of the barrier function of vaginal mucosal epithelium was performed as previously described , 18, 19.software .stratum corneum in ectocervical epithelium was quantified using NDP.view2 software (Hamamatsu Photonics K.K.). For DSG1 immunohistochemistry, unstained sections were de-paraffinized in xylene and sequentially rehydrated with 100% ethanol, 96% ethanol, and deionized water. Antigen retrieval was performed in citrate buffer with 0.02% Tween 20 (pH = 6.0) for 20 min at 95°C. Endogenous peroxidase activity was quenched in 3% hydrogen peroxidase and sections placed for 4 h with 5% normal goat serum at 4°C  and 24 h at 4°C in rabbit anti-DSG1 (clone EPR67766(B), Abcam Inc, Cambridge, MA) diluted 1:100 with SignalStain® antibody diluent . Using manufacturer’s instructions, sections were washed thrice and incubated with SignalStain® Boost detection and SignalStain® DAB Chromogen . Sections were counterstained with hematoxylin and placed in mounting medium . For DSG1 protein quantification, images were captured using the NanoZoomer 2.0-RS slide scanner and analyses performed using ImageJ software [Ectocervical tissue was fixed for 48 h in 4% formaldehyde before paraffin embedding. Five μm sections were stained with hematoxylin and eosin (H&E) and imaged with the NanoZoomer 2.0-RS . Thickness of the software .DSG1, Rh02840468_m1), desmocollin 1 , occludin , tight junction protein 1 , claudin 1  and the housekeeping gene ribosomal protein L32  [RNA was isolated from ectocervical tissue using the RNeasy Lipid Tissue Kit and RNase-free DNase Set (Qiagen) using manufacturer’s instructions. Samples were re-suspended in nuclease-free water to quantify RNA via spectrophotometer . 100 ng of RNA were used to generate cDNA using the SuperScript™ IV VILO™ Master Mix . For reactions, TaqMan™ Fast Universal PCR Master Mix (Applied Biosystems®) was used in combination with cDNA template and specified primers. Gene expression levels for desmoglein-1  was used for statistical analyses. Where indicated, normal distribution was tested by D'Agostino & Pearson omnibus test or evaluation of the residuals . The paired Student Macaca nemestrina) that 2–3 weeks earlier had been IM administered 15–20 mg of DMPA [Prior publications identified serum MPA levels of 21—25 nM in pigtail macaques  and 9.4 pg/mL (34.5 pM) before and after DMPA treatment (4 levels of 847 pg/mL (2694 pM) before treatment and 70 pg/mL (98 pM) after treatment  axis . Prior p.9 pg/mL . Using breatment . In DMPAreatment . Togethestratum corneum thickness was significantly reduced in RM administered DMPA or DMPA and Depo-E compared to untreated controls -containing compound. In analyses of H&E-stained ectocervical sections, controls . Quantif animals . As oppoand OCLN . CongruePA alone . When coe and RM . Moreoverogestin .2 concentrations measured at the time of vaginal biopsy collection (Because treatment of RM with DMPA significantly reduced genital levels of the desmosomal cadherins DSG1 and DSC1, we posited that treatment similarly compromises genital mucosal barrier function. As hypothesized, vaginal biopsies from untreated and DMPA-treated RM showed ex vivo penetration of LMM molecules into the vaginal epithelium was significantly enhanced by DMPA treatment . Moreovellection , resultsllection .Clinical investigation faces considerable obstacles to illuminate a clear path forward regarding the use of DMPA in areas of the world where large numbers of women are living with HIV, an observation that implies alternative research strategies will be needed to help define this relationship. Whereas multiple reports in the past 35 years showed enhanced HIV risk in women using DMPA, all of these data were acquired from observational studies , 10. Rep2 levels induced by DMPA vs. the normal menstrual cycle and a stronger agonistic effect of MPA vs. endogenous progesterone on the progesterone receptor [While performing more clinical trials will help define actual relationships between HIV and individual forms of contraception, the ability to appropriately model changes in antivirus immunity produced by these contraceptives can clarify these links and provide findings unobtainable in clinical research. Our current results show that RM model important elements of the human host response to DMPA and establish this progestin significantly reduces genital levels of the desmosomal cadherins DSG1 and DSC1 and impairs genital epithelial barrier function. Moreover, these effects are produced in RM with pharmacologically relevant serum levels of MPA. In support of their clinical relevance, current findings mirror the lower DSG1 levels and compromise of genital epithelial barrier function generated in women initiating DMPA or LNG-IUS use , 19, 31.receptor . Unlike receptor , we alsoreceptor  as well receptor . Currentreceptor  and highreceptor .stratum corneum. Together, these animal model findings challenge the notion that the stratum corneum is an all-important component of the genital epithelial barrier [stratum corneum or overall genital epithelial thickness. While we are currently exploring the latter possibility in RM SIV challenge studies, our current findings already establish that DMPA-mediated changes to genital epithelial integrity and barrier function are reversed by concomitant administration of E-containing compounds. These results are congruent with mouse model data that showed combined treatment with progestin and E avoided the compromise of genital epithelial barrier function and uniform susceptibility to genital HSV-2 or HIV-1 infection produced by DMPA treatment alone [stratum corneum thickness, exogenous E prevented DMPA-mediated increases in genital epithelial permeability. These findings may result from partial inhibition of E-mediated epithelial proliferation and differentiation induced by strong agonistic effects of MPA on the progesterone receptor [DSG1 expression, desmosome organization, and maintenance of genital epithelium integrity in mice, RM, and humans [Also congruent with our mouse model findings , current barrier , 40 and nt alone , 13, 18.receptor , 42. Altd humans , presumad humans , 47.In summary, results from the current investigation identify the RM as an appropriate preclinical model for exploring the effects of sex steroids on genital epithelial barrier function. Our findings also imply that RM studies are well-positioned to illuminate sex steroid-mediated effects on genital pathogen susceptibility and help delineate contraceptive strategies least likely to promote genital infection. As significant hurdles are in place that will continue to impede clinical studies from defining precise relationships between HIV susceptibility and contraception, RM and mouse models seem indispensable for informing both clinical study design and recommendations for contraceptive choice in regions with large HIV burden and for providing women worldwide the type of data needed to make reasoned decisions regarding contraceptive selection.