Avian H3N2 influenza virus follows cross-host transmission and has spread among dogs in Asia since 2005. After 2015–2016, a new H3N2 subtype canine influenza epidemic occurred in dogs in North America and Asia. The disease prevalence was assessed by virological and serological surveillance in dogs in China. Herein, five H3N2 canine influenza virus (CIV) strains were isolated from 1185 Chinese canine respiratory disease samples in 2017–2018; these strains were on the evolutionary branch of the North American CIVs after 2016 and genetically far from the classical canine H3N2 strain discovered in China before 2016. Serological surveillance showed an HI antibody positive rate of 6.68%. H3N2 was prevalent in the coastal areas and northeastern regions of China. In 2018, it became the primary epidemic strain in the country. The QK01 strain of H3N2 showed high efficiency in transmission among dogs through respiratory droplets. Nevertheless, the virus only replicated in the upper respiratory tract and exhibited low pathogenicity in mice. Furthermore, highly efficient transmission by direct contact other than respiratory droplet transmission was found in a guinea pig model. The low-level replication in avian species other than ducks could not facilitate contact and airborne transmission in chickens. The current results indicated that a novel H3N2 virus has become a predominant epidemic strain in dogs in China since 2016 and acquired highly efficient transmissibility but could not be replicated in avian species. Thus, further monitoring is required for designing optimal immunoprophylactic tools for dogs and estimating the zoonotic risk of CIV in China. Orthomyxoviridae that have a complex natural history with a wide range of host species [Influenza A viruses (IAVs) are segmented single-stranded RNA (ssRNA) viruses belonging to  species ,2. Based species . Wild wa species ,5. Occas species , equines species , seals [ species , dogs [9 species , civets  species , nonhuma species , chinstr species , martens species ,15,16. S species . The can species .In 2005–2006, the avian-origin H3N2 was first reported in canines in Asia, including South Korea and China, and later acquired the capability of dog-to-dog transmission ,20. The In order to determine the circulation dynamics in China, virological surveillance and seroepidemiological investigation of CIVs were carried out in this study. Chinese families are exposed to a large number of pet dogs that may serve as intermediate hosts for transmitting the influenza virus to humans.With the increasing number of pet dogs in China, whether the canine could serve as an intermediate host for transmitting the influenza virus to humans is a major concern. The reassortment events between CIVs and other influenza viruses, including the H5N1 avian virus and the human H1N1 pandemic virus, have occurred ,27. SeveA total of 1185 nasal and throat swabs were collected from dogs with respiratory symptoms in veterinary clinics or kennels in Jilin, Henan, and Guangdong provinces in China to monitor the H3N2 CIV epidemic and virus evolution in January 2017–May 2019. Sample processing and virus isolation were conducted as described previously . Embryonwww.ncbi.nlm.nih.gov accessed on 5 July 2021) and the GISAID (www.gisaid.org accessed on on 5 July 2021) database. Consensus sequence editing, alignment, and phylogenetic analysis were performed with Mega7.0. Total sequence alignment lengths (in nucleotides (nt)) were as follows: PB2, 2122; PB1, 2134; PA, 2048; HA, 1598; NP, 1394; NA, 1334; M1, 727; NS1, 655. Phylogenetic analyses were carried out for all eight gene segments using the maximum-likelihood (ML) method with 1000 bootstrap replicates, the general time-reversible (GTR) substitution model, and gamma-distributed rate variation among sites.To characterize the evolution of CIV H3N2, we sequenced the full genome of five strains and performed genetic analyses using H3N2 virus sequences of human, avian, and canine species from GenBank . Briefly, 25 μL of serial two-fold dilutions of serum samples were mixed with four units of hemagglutination titer of the virus (A/canine/Jilin/QiaoKe01/2017 (H3N2) CIV strain (GenBank accession no. MZ948848-MZ948855)) in V-shaped microtiter plates and incubated at room temperature for 30 min. Then, 25 μL of 1% chicken red blood cells (RBCs) were added to each well and incubated at room temperature for 40 min. The HI titer was expressed as the reciprocal of the highest serum dilution that completely inhibited the hemagglutination of the virus. Serum samples with up to 8 antibody titers were considered positive. The animal experiment was approved by the Institutional Review Board (or Ethics Committee) of Key Laboratory of Jilin Province for Zoonosis Prevention and Control .A total of 3197 serum samples fom clinically healthy pet dogs were collected across 27 provinces in China, of which 913 were collected from January to December in 2017 and 2284 were collected from January to December in 2018. In addition, 208 serum samples were collected from pet dogs with clinical symptoms of respiratory disease in veterinary clinics in 2018, 130 serum samples were collected from stray dogs in Jilin in 2017, and 44 serum samples were collected from Chinese rural dogs in Shannxi in 2018. The hemagglutination inhibition (HI) test was used to detect the serum samples. The test was performed according to procedures recommended by the World Organization for Animal Health (OIE) (n = 4), contact (n = 3), exposed (n = 3), and control (n = 1) groups. Dogs in the inoculated group were anesthetized with ketamine and inoculated intranasally with 107 EID50 (50% egg infections dose) in a volume of 1 mL and placed into the contact (same cage) and exposed (1 m distance) groups after 24 h. All dogs were monitored daily for temperature, physical activities, and nasal/throat swabs. At 3 days post-infection (dpi), one dog from the inoculated group was euthanized, and each lung lobe , lung lymph nodes, spleen, intestine, mesenteric lymph nodes, and trachea were taken for hematoxylin–eosin (H&E) staining and immunohistochemistry (IHC) with mouse antiserum against the QK01 virus and goat anti-mouse IgG as the secondary antibody . The nasal and throat swabs of each dog were mixed to determine the EID50 by injection of chicken embryos. The venous blood of each dog was collected to determine HI antibody levels at 14, 21, 28, and 35 dpi.A total of 11 beagles (4 months old) that tested negative for serological antibody against H3N2 CIVs were chosen from the Rui man dog training field, Shenbei New Area , and assigned randomly to four groups, including inoculated  with the diluted virus solutions in a volume of 50 μL, and the control group was inoculated with an equivalent volume of phosphate-buffered saline (PBS). At 3 dpi, three mice in the 106 EID50 group were sacrificed, and organs  were collected for viral RNA load analysis. The animal body weight was monitored daily until 14 dpi.A total of 30 BALB/c mice  were purchased from the Beijing Vital River Laboratory  and randomly divided into six groups. The virus solution was diluted to a range of 10n = 3), contact (n = 3), and exposed (n = 3). The six guinea pigs in the inoculated group were anesthetized with ketamine and inoculated intranasally with 2 × 106 EID50 in a volume of 200 μL; of these, three guinea pigs were placed on the left side of three cages individually. After 24 h, each of these animals was placed in a cage with one uninfected guinea pig. The other uninfected guinea pig was placed in a nearby cage (5 cm apart) as the exposed group . Then, they were euthanized on days 3, 5, and 7 post-inoculation individually, and the lungs and tracheas were collected for H&E and IHC staining to observe pathological changes and virus replication.Totally 12 Hartley female guinea pigs  were used to assess the transmission capacity of the virus, as described previously (13). All animal experiments were carried out in special cages. Each cage had two independent spaces on the left and right sides separated by 5 cm. The animals were randomly divided into three groups: inoculated (ed group . The nasn = 7), contact (n = 3), exposed (n = 3), and control (n = 2). Chickens in the inoculated group were infected intranasally with 3 × 106 EID50/0.1 mL and placed into the contact (same cage) and exposed (0.5 m distance) groups after 24 h. Throat and cloacal swabs were collected daily to detect virus titers in 9-day-old chicken embryos. Venous blood samples in all groups were collected to determine HI antibody levels on 14 and 21 dpi. Furthermore, 11 domestic ducks (9 weeks old) were purchased from Changchun Hao Yi animal farm  and randomly divided into four groups: inoculated (n = 3), contact (n = 3), exposed (n = 3), and control (n = 2). The experimental protocol was the same as the above chicken experiment.In order to assess whether the virus can infect poultry after long-term adaptation in dogs, we chose SPF chickens and domestic ducks for infection. All animals were seronegative for H3N2 CIV. A total of 15 SPF chickens  were purchased from Harbin Weike Biotechnology Co., Ltd.  and randomly divided into four groups: inoculated (n = 397), Liaoning (n = 616), Henan (n = 24), Jiangsu (n = 50), and Guangdong (n = 98) in 2014–2019. The 1185 swab samples consisted of 5 positive nucleic acids across Jilin (1), Henan (3), and Guangdong (2).Five strains of the H3N2 influenza virus (0.42%) were isolated from 1185 swab samples from canines. Swab samples from dogs were collected in animal hospitals in four provinces, including Jilin (The isolated strains in this study were termed A/canine/Jilin/QiaoKe01/2017 (QK01(H3N2)), A/canine/Henan/L02/2018 (L02(H3N2)), A/canine/Henan/L03/2018 (L03(H3N2)), A/canine/Henan/L06/2018 (L06(H3N2)), and A/canine/Guangdong/GY01/2018 (GY01(H3N2)). The sequences of these isolates were submitted to GenBank at NCBI (GenBank accession MZ948848-MZ948855 and OK354170-OK354201), and A/canine/Jilin/QiaoKe01/2017 (QK01(H3N2)) was selected for further characterization.In order to determine the evolution of H3N2 CIV in 2017–2019, we performed a phylogenetic analysis of eight segments. Each genome segment of the five isolated H3N2 CIVs shared a high nucleotide sequence identity. The phylogenetic analysis of HA and NA indicated that H3N2 strains isolated from dogs are independent evolutionary branches in the phylogenetic tree, which is far from H3N2 in humans and swine . The phyPhylogenetic tree of H3N2 CIV internal sequences.In this study, no virus belonging to the clade before 2016 was detected, suggesting that the new antigenic variation lineage arisen in 2017 was dominant in the dog population.A total of 3579 dog serum samples from 27 provinces in China were screened by the HI assay for antibody reactivity against A/canine/Jilin/QiaoKe01/2017 (QK01(H3N2)). As shown in 50 of chicken embryos, and infectious virus particles could be detected in beagles in all groups except the control group.The replication and transmission of the virus in beagles were assessed in the contact and exposed groups at 1 dpi and thereafter. The results showed that beagles in the inoculated, contact, and exposed groups had typical respiratory symptoms, including sneezing, watery eyes, coughing, and shortness of breath. As shown in Furthermore, HI antibodies in each dog were detected at 14, 21, 28, and 35 dpi. At 14 dpi, HI antibodies were observed in the inoculation (3/3), contact (2/3), and exposed (3/3) groups. At 21 dpi, all experimental dogs showed high HI antibody titers (32–64). At 35 dpi, the exposed group (256) showed higher HI antibody titers than the inoculated (32–64) and contact (64) groups. These results suggested that natural infection with H3N2 CIV may contribute to the expansion of memory B cells, enabling the production of more antibodies. The above findings indicated that the H3N2 CIV could efficiently be transmitted via contact and respiratory droplets.Subsequently, histological assays were conducted to assess pathological changes in virus-infected dogs at 3 dpi . H&E res2–106 EID50 of the virus, respectively. As shown in 6 EID50 of influenza virus were killed on 3 and 5 dpi, and their organs, including nasal turbinate, lung, spleen, kidney, and brain, were harvested for viral titer detection. Viral replication was detected in the nasal turbinate and lung of mice but not in the spleen, kidney, or brain (To evaluate the replication of viruses in mammals, we inoculated groups of five mice i.n. with five diluted solutions 10or brain . These rGuinea pigs have been used as animal models to evaluate the replication and transmission of the influenza virus. Nasal washes and serum samples were obtained to detect the virus and HI antibody titers. As shown in Furthermore, SPF chickens and domestic ducks were chosen to assess whether the virus could infect poultry after long-term adaptation in dogs. As shown in H3N2 CIV, a pathogen that causes severe and acute respiratory disease in dogs, has been circulating in China for more than a decade. However, the prevalence and outbreak mode of this virus remains to be further studied. The emergence of the H3N2 CIV and its evolutionary and epidemiological patterns with multiple, periodic introductions in the USA were described by Voorhees et al. . In the Animal studies demonstrated that elevated body temperature, virus shedding, and seroconversion were detected in infected and contacted beagles. Experimental infections in mice, guinea pigs, and beagles provided insights into the pathogenicity of these viruses in mammals. Mild histopathological changes, including hyperplasia of pneumocytes and infiltration of lymphocytes, were recorded in guinea pigs and beagles infected with the QK01 isolate. Furthermore, the transmission experiment showed that the QK01 isolate could be transmitted to naïve beagles via respiratory droplets, which suggested that currently circulating strains might be readily transmitted among mammalian hosts. Dog-to-dog transmission through respiratory droplets of the circulating canine virus might cause a rapid spread of the virus in a short period in the dog population. Considering the large number of pet dogs and the increased probability of contact between humans and pets, there is a potential risk of virus transmission from dogs to humans.Taken together, these results suggested that currently circulating H3N2 CIVs antigenic variants might have entered China from the USA. Furthermore, these viruses are continuously evolving in canine species, have expanded the host range, and might be established in pet cats. Importantly, these findings underscore the importance of continuous surveillance to understand the genetic diversity and zoonotic risk of CIVs in China.


These will be beneficial for further studies to a better understanding of PEDV biology and PEDV vaccine development.Porcine epidemic diarrhea virus (PEDV) is a causative agent of a highly contagious enteric disease in swine of all ages, leading to severe economic losses for the swine industry in many countries. One of the most effective approaches in controlling PEDV infection is vaccination. The ORF3 accessory protein has been proposed as a crucial viral virulence factor in a natural host. However, due to the lack of an extensive comparative study of ORF3, exactly how the ORF3 takes part in virus replication and pathogenesis as well as its role in host-virus interaction is unclear. In this review, we aim to discuss the current knowledge of ORF3 concerning its dispensability for viral replication  Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen that has spread in the swine population. PEDV infection causes severe watery diarrhea, dehydration, vomiting, and death, particularly in neonatal piglets, resulting in massive economic losses in pig industries worldwide, particularly in the United States, China, South Korea, and Thailand –4. AccorAlphacoronavirus, PEDV is an enveloped virus bearing positive-sense single-stranded RNA of approximately 28 kb in length. The viral genome comprises at least seven open reading frames ] is a viroporin, consisting of four transmembrane domains possessing a potassium ion channel activity  Figure . Viral pactivity , which mactivity . Howeveractivity , virus ractivity . Neverthactivity , 21. Theactivity . Howeverin vivo model. While the function of ORF3 remains to be determined, mounting evidence points to its strong association with viral virulence. As PEDV targets intestinal epithelial cells for productive infection in vivo, this process may require the functional ORF3, possibly via its ion channel activity. However, most in vitro studies are based on cultured cells such as Vero, MARC-145, and IPEC-J2, which may not be biologically relevant to what happens in vivo. As organoid systems, especially 3D intestinal organoids, could be highly relevant models for assessing differences between PEDV variants, future studies using this platform are needed to gain more insights into the functional role of ORF3.Wild-type PEDV and cell culture adapted PEDV (ca-PEDV) have been shown to exert different degrees of disease severity in the infected hosts. Bernasconi et al.  showed tin vitro  and CV777 (bearing the full-length ORF3), were reported to grow more efficiently in Vero cells stably expressing ORF3 than those propagated in parental Vero cells . MoreoveBesides its direct role in PEDV replication, accumulating evidence has suggested that ORF3 might play a role in several cellular processes. It has been shown that ORF3 could regulate the cell cycle progression by prolonging the S phase , inhibitIn addition, deletion or truncation of the ORF3 acquired during propagation in cell culture has been used as a genetic marker to differentiate between the field and cell-adapted strains. However, the recombination of a highly pathogenic PEDV and a low pathogenic vaccine strain , and a nEven though the underlying mechanisms of ORF3 functions related to pathogenicity, virus adaptation, and virus proliferation have not been elucidated, several lines of evidence have emphasized the impact of the interplay between ORF3 and the host's machinery. Microscopic inspection of cells over-expressing the ORF3 showed that the protein was typically localized in the cytoplasm , 34, 36,170YLAI173) is essential for the translocation of ORF3 from the ER to the plasma membrane  of amino acid sequence responsible for this particular aspect can help overcome difficulties in PEDV isolation in vitro and in vivo. The mechanistic role of ORF3 in interferon signaling pathway/inflammasome activation could be further explored. Knowledge obtained from these studies would be a groundwork for viral gene mutagenesis to improve a better live-attenuated PEDV vaccine accomplishing high yield in cell culture with a minimum passaging, induction of specific immune response, and no reversion to virulence in the vaccinated animal.ORF3 is a multi-functional protein that plays essential roles in modulating cellular mechanisms, particularly the host immune system and apoptosis. These roles of ORF3 are likely associated with virus replication and pathogenicity. Due to the lack of comprehensive studies of ORF3 and inconsistent findings of ORF3 among available literature, its impacts on virus adaptation in cell culture, virus replication, and pathogenesis are still not well-elucidated. Future studies utilizing various approaches are needed to gain more precise insights into our understanding of this protein. For example, a series of PEDV ORF3 mutations (variants) should be constructed together with the S protein to generate recombinant PEDVs to show the effect of each mutation in the virus phenotype. The impact of ORF3 variants on PEDV growth kinetics should be investigated in detail All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.This work was supported by National Vaccine Institute (NVI), Thailand, under grant number P2150344.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aim of the study was to investigate the issue of medial midvastus (MMV) vs. medial parapatellar (MPP) approaches in total knee arthroplasty (TKA). It was hypothesized that the two surgical approaches would produce significantly different results with respect to patient-reported knee score outcome (hypothesis 1), short-term postoperative range of motion (ROM) (hypothesis 2), long-term postoperative ROM (hypothesis 3) and prosthesis survival (hypothesis 4).A retrospective comparative study design was applied. Data sets were obtained from the state arthroplasty registry. The Western Ontario and McMaster Universities osteoarthritis index (WOMAC) data were analyzed from preoperative and 1 year postoperatively. The ROM data were analyzed for the time points preoperative, postoperative days 4 and 10 and 1 year.p = 0.305 and p = 0.383, respectively, hypothesis 2). Likewise, ROM did not significantly differ between the groups 1 year after TKA . The 5‑year prosthesis survival did not differ between the groups and showed 94.46%  in the MMV group and 94.33% (95% CI 89.96–96.83%) in the MPP group .Available were 627 cases (407 MMV vs. 220 MPP) and 1 year postoperatively there were no significant differences between groups regarding the WOMAC scores (hypothesis 1). Early postoperatively on days 4 and 10 after TKA there were no differences between groups (Both surgical approaches produce equivalent clinical results in terms of early postoperative ROM, late postoperative ROM and 1‑year WOMAC. The same prosthesis survival rates can be expected. Up to 30% of patients were reported to dissatisfied with the outcome of total knee arthroplasty (TKA) because of unexplained pain , 15. WheRegarding surgery-related factors previous researchers discussed the role of different surgical approaches on outcome after TKA , 13, 18.Due to the abovementioned conflicting evidence and the rather small sample sizes it was the aim of the study to investigate the issue of MMV vs. MPP approach in TKA once again but on the basis of much larger study populations. It was hypothesized that the two surgical approaches would lead to significantly different results with respect to patient-reported knee score outcome (hypothesis 1), short-term postoperative ROM (hypothesis 2), long-term postoperative ROM (hypothesis 3) and prosthesis survival (hypothesis 4).A retrospective comparative study design was applied. Data available from clinical routine were analyzed after approval by the IRB of the authors’ affiliated institutions. Patients who previously underwent primary TKA as part of the clinical routine were analyzed. Data sets were obtained from the state arthroplasty registry and covered all primary TKA cases performed at Department of Orthopaedics and Traumatology, Medical University of Innsbruck, Innsbruck, Austria from 2008 to 2015. Cases were excluded in the case of a) surgical approaches other than MPP and MMV, b) implants other than Scorpio CR or Triathlon CR , c) incomplete preoperative WOMAC data and d) incomplete postoperative WOMAC data (1 year)   was avaiThe ROM data collected with goniometers during clinical routine were taken from the medical records for the following points in time: preoperative, postoperative days 4 and 10 and 1 year.As descriptive measures for WOMAC scores and ROM at different time points we estimated mean value and standard deviation. Differences between groups were tested for preoperative and postoperative WOMAC scores applying the Mann-Whitney U-test and for ROM applying the t‑test. Prosthesis survival was estimated with the Kaplan-Meier method from date of surgery until date of revision, data of death or end of follow-up (31 December 2015), whichever occurred first. We report Kaplan-Meier estimates for prosthesis survival at 1–5 years together with 95% confidence intervals (CI). Differences in survival curves were tested using the log-rank test. Data analysis was performed with Stata Version 13 . Alpha was defined as 0.05 (two-tailed tests).Available for analysis were 627 cases (407 MMV vs. 220 MPP). In the MMV group the mean age was 70.1 ± 9.1 years, 64.1% of the participants were female and in 43.7% the left side was operated. In the MPP group the mean age was 69.0 ± 9.7 years, 57.7% of the participants were female and in 45.5% the left side was operated. None of these demographic parameters differed significantly between the groups. Preoperative leg axis (mFTA) from whole leg radiographs was also similar between the groups . Early postoperatively on days 4 and 10 after TKA there were also no differences between groups  in the MMV group and 94.33% (95% CI 89.96–96.83%) in the MPP group , short-term postoperative ROM (day 4 and day 10), long-term postoperative ROM (1 year) or prosthesis survival.For the purpose of comparing these findings with those of previous researchers a comprehensive literature analysis was performed. The search term “total AND knee AND (arthroplasty OR replacement) AND midvastus” produced 94 matches. After excluding irrelevant studies and those comparing approaches other than MMV and MPP there remained 15 original research articles , 23, 25.Regarding sample size most of the previous studies recruited around 50 patients for both groups together. Only 2 of the previous studies reported much higher sample sizes of around 200 for both groups together , 25. TheThe following limitations of the study are acknowledged. The outcome parameters were predominantly mid-term . Only few parameters dealt with early postoperative function (day 4 ROM and day 10 ROM). This must be regarded as a limitation because potential early postoperative benefits of the MMV approach might therefore have been overlooked. Parameters, such as SLR, stair climbing, Q‑strength would have been of additional value. Also, it would have been beneficial to include knee score data, such as the WOMAC from 3 months postoperative. Other limitations are the facts that the patients were operated on by a large variety of surgeons and that no outcome parameters other than those mentioned above could be collected due to the retrospective nature of the study. The following facts are regarded as strengths of the study. It was the largest study conducted to date with an overall sample size of more than 600. Data quality is regarded as very robust as data originate from the state arthroplasty registry. Another advantage was that for the first time the question of the surgical approach in TKA was linked to prosthesis survival as reported from an arthroplasty registry.The clinical relevance of the findings is regarded as high. Both surgical approaches may be used in daily practice during TKA. Using the MMV approach does not negatively affect prosthesis survival in the long run.On the basis of the findings, it is concluded that both surgical approaches in primary TKA, MMV and MPP, produce equivalent clinical results in terms of early postoperative ROM, late postoperative ROM and knee score outcome. The same prosthesis survival rates can be expected when using the MMV or the MPP approach in primary TKA. Consequently, the question whether to perform MMV or MPP depends on the surgeon’s preference.


Allexivirus, Carlavirus and Potyvirus genera followed by virus-specific colorimetric dot-blot hybridization. Assays targeted the potyviruses (onion yellow dwarf virus (OYDV), shallot yellow stripe virus (SYSV), and leek yellow stripe virus (LYSV)), the carlaviruses (garlic common latent virus (GCLV) and shallot latent virus (SLV)), and the allexiviruses  and shallot virus X (ShVX)). Virus incidence in crops was consistently high, with most plants infected with at least one virus from each genus. OYDV, LYSV, SLV, and GCLV were commonly detected. Three of the four allexiviruses were in all districts surveyed but varied in incidence, whereas ShVX and SYSV were not detected. There was no association between virus species complement and bulb size, indicating size is not a good predictor of the virus status of planting material. The variation of virus incidence across different Australian growing districts and in different cultivars implies multiple introductions of viruses rather than spread within the country. The genetic diversity observed within coat protein sequences of some virus species also supports multiple separate introductions.The distribution of viruses in eastern Australian field garlic was evaluated. Detection assays were developed that involved generic RT-PCR for viruses in the  Allium sativum, is a member of the Amaryllidaceae plant family and is a close relative of onion (A. cepa), shallot (A. cepa var. aggregatum), leek (A. ampeloprasum), and chive (A. shoenoprasum). Commercially, garlic is cultivated through vegetative propagation and is both day-length and temperature sensitive. Garlic planting windows and seasons vary considerably around Australia, and consequently a wide range of cultivars are grown. Cultivars are often locally adapted based on performance under specific day-length and temperature conditions. Australia imports around 10,000 tonnes of garlic per year, mostly sourced from China, but with increasing amounts sourced from Spain, Argentina, Mexico, and the USA. Currently, the local production of garlic in Australia is about 500 tonnes per annum, spread across different climatic zones from subtropical south-east Queensland to temperate Victoria and South Australia [Garlic , onion yellow dwarf virus (OYDV), and shallot yellow stripe virus (SYSV) [shallot latent virus (SLV—syn. garlic latent virus) and garlic common latent virus (GCLV) [Iris yellow spot virus (IYSV) [There are at least twelve different virus species that infect alliums. Of these, the most common are the potyviruses, s (SYSV) ; the cars (GCLV) ; the alls (GCLV) ,12,13; as (IYSV) ,15. The s (IYSV) .lettuce necrotic yellows virus (LNYV) was reported from diseased field garlic from central Victoria [Globally, numerous studies have identified and characterized the viruses infecting garlic and established their effect on yield ,18,19,20Victoria . The preVictoria ,26. ThesVictoria .Potyvirus, Carlavirus, and Allexivirus, a novel molecular diagnostic method was developed. The new method aimed to detect all 10 target viruses using RT-PCR with a single reverse transcriptase reaction, primed with an oligo-dT and then three genus-specific forward primers in PCR. Individual virus species were then identified from the RT-PCR amplicons with specific probes using a dot blot hybridization assay. The distribution of virus species within planting propagules was also examined to investigate the hypothesis that all cloves from an infected bulb are uniformly infected.The aim of this study was to determine the identity and distribution of viruses in eastern Australian commercial garlic crops. To efficiently assess disease survey samples for viruses in the genera The method developed in this study allowed efficient indexing of over 3500 field plant samples and was used to evaluate trial plants. The genus-level degenerate RT-PCR successfully amplified the expected viruses, and individual probes identified the specific viruses. The results from up to 100 selected samples per virus were verified using specific RT-PCR for GarVA, GarVB, GarVC, GarVX, GCLV, SLV, OYDV, and LYSV and confirmed the accuracy of the probe-based assay.Indexing for IYSV was done separately using a previously published assay , as it iThis work represents a large-scale evaluation of virus distribution across several of Australia’s main garlic producing regions . The genVirus incidence detected by genus-specific RT-PCR within the surveyed crops ranged from 90 to 100% across individual samples from Queensland sites . IncidenSpecies-specific virus testing showed that plants were chronically infected with the potyvirus OYDV, the carlavirus SLV, and the allexiviruses GarVA, GarV-B, and GarV-X . High inProfiles of the allexiviruses varied distinctly across growing districts. Crops tested from districts in South Australia and New South Wales displayed lower virus incidences than those from Queensland and Victoria, particularly at Renmark, South Australia, where only very low levels (<8% of GarVX) were detected. In Queensland, GarVA, -B, and -X were present at high levels, >50%, throughout most crops. In Victoria, GarVX was more commonly detected than GarVA and GarVB. GarVA was generally present in the Queensland crops tested, while in material surveyed from southern Australia, the virus was only detected on two Victorian properties . These iOnly the Queensland surveyed crops were tested for the tospovirus IYSV, and it was not detected in any samples.The incidence of viruses of individual species within cloves of field grown bulbs was evaluated through indexing plantlets derived from viable cloves. A comparison of virus incidences was made between planting material selected as either large bulbs (>80 g) or small bulbs (20–30 g) . The aveVirus incidence within the clove material was very high, with all six viruses detected  sequences for the selected viruses detected in the surveyed Australian garlic were compared with isolates from GenBank.The potyviruses clustered in three well-supported clades, representing OYDV, SYSV, and LYSV . OYDV CPFor the carlavirus sets, GCLV CP nucleotide sequences were 88–99.6% identical, and for SLV they were 78.1–99% identical . The GCLThe allexiviruses formed two well-supported clades on the phylogenetic tree, with one containing GarVA and GarVD, and the other GarVB, GarVC, and GarVX . NucleotThis study presents the first comprehensive virus survey of Australian garlic crops. Surveying of a range of garlic cultivars from plantings across four Australian States ensured a widespread assessment of the virus status of crops comprising a range of commercial cultivars across the industry. Symptoms of virus infection were present in all plants, and virus presence was confirmed by molecular diagnostic testing. Virus-free planting material was distributed to some Victorian growers in the 1990s  and may Molecular testing showed that Australian field-grown garlic cultivars are chronically infected with a complex of viruses, with more than 90% of plants infected with viruses from one or more species from each of the three genera tested. No single infections were recorded. Probe hybridization assays showed that the potyvirus OYDV and the carlavirus SLV were the most widespread viruses found in the surveyed crops. This result contrasts starkly with that of Sward , who faiAlthough a limited number of samples were examined for virus diversity, phylogenetic analysis of partial or complete CP sequences from this work and that of Wylie  revealedThe allexivirus ShVX and the potyvirus SYSV were not reported in the current study of surveyed field garlic nor in any of the previous studies, so it is unlikely that these viruses are present in Australian garlic. IYSV, a tospovirus, is known to infect alliums such as onions, chives, leeks, and garlic, and in Australia it has been recorded from onion crops in WA, NSW, and Victoria . In the The evaluation of virus distribution within large and small bulb planting material showed that none of the plantlets that emerged from the cloves were virus free, and almost all were found to have mixed infections, predominantly comprising OYDV, LYSV, SLV, and one or more of the three allexiviruses: GarVA, GarVB, and GarVX. Plantlets derived from cloves from large bulbs were almost uniformly infected with all six viruses. By contrast, the virus incidence in plants derived from cloves from small bulbs was variable. In most instances where there were non-uniform virus infections, it was due to an absence of one or more of the allexiviruses. No correlation was observed between reduced clove size and virus complement. Instead, clove size varied independently of infection status in this experiment.These results highlight two key aspects of garlic virus disease. First, infection of garlic with viruses from multiple species per se doesn’t necessarily correlate well with yield reductions, as the bulbs in this study were all from a single cultivar sourced from one property and were infected with the same range of virus species, irrespective of their size. Thus, the selection of planting material based on size or quality is unlikely to significantly reduce virus incidence in subsequent crops. However, the titer of individual viruses may be an important factor in determining the impact of viruses on yield as opposed to simply the presence of the virus, and selective gene silencing may play a role. The second point is that bulb size is not a good indicator of virus status for use as planting material, as cloves from the smaller bulbs sometimes contained fewer viruses than cloves from the larger bulbs. However, bulb size is a good indicator of yield in the subsequent crop, and research has shown that the consistent re-selection of the largest bulbs with more vigorous blue-green foliage  provides substantial generational yield improvement .The high incidence of viruses in garlic cultivars across all Australian growing areas and the recorded yield benefits of growing virus-free garlic ,3,4,5,7 This study evaluated the distribution of viruses in eastern Australian field garlic and provided new methodology for multiplex detection of 10 individual virus species. The variable distribution of individual virus species across the different growing districts and cultivars implies multiple introductions of viruses into Australia rather than spread within the country. The genetic diversity observed within coat protein sequences of some virus species also supports multiple independent introductions. Evaluation of planting propagules indicated bulb size was not a good predictor of the virus status of planting material; however, it has potential as a predictor of subsequent yield.To efficiently test for the 10 virus species, molecular indexing involved RT-PCR amplification using genus-specific primers for allexiviruses, carlaviruses, and potyviruses. This was coupled with species-specific dot blot hybridization assays  to identify the individual virus species. IYSV was tested using specific primers for the virus, as described by . DetailsTotal nucleic acid was extracted from leaf tissue samples using the Biosprint DNA Plant Kit, according to the manufacturer’s instructions , though omitting the RNAse from the buffer RPW. The synthesis of cDNA was carried out using the reverse transcription enzyme Superscript III  and the universal Poty1 reverse primer  accordin2, 100 µM dNTPs, 2.0 µL cDNA, and 0.2 U Taq Polymerase  in a total volume of 25 µL. The PCR cycle parameters were as follows: initial denaturation 94 °C for 1 min, then 35 cycles of 94 °C for 20 s, 56 °C annealing for 60 s, 72 °C extension for 60 s, and a final extension at 72 °C for 3 min. For IYSV, PCR cycle conditions were as follows: 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 48 °C for 30 s, and 72 °C for 45 s, followed by a final extension at 72 °C for 5 min.Genus level PCRs contained 10 pmol degenerate forward primer corresponding to the virus genus being amplified . The cycling conditions included an initial denaturation at 95 °C for 1 min, followed by 25 cycles of 95 °C for 15 s, 56 °C for 20 s, 72 °C for 40 s, and a final extension step of 72 °C for 5 min. For GarVC, the annealing temperature was adjusted to 50 °C.For dot-blot hybridization assays, probes were generated through PCR amplification of approximately 1 ng/µL of recombinant plasmid from selected virus isolates sourced from Australia or introduced under permit from overseas . Specifi2O) and applied to Hybond-N+ membrane (Roche) in 1 µL volumes using a grid pattern (2) (Roche) at 42 °C for 30 min with rotation. Excess hyb-solution was removed, and the denatured virus-specific digoxigenin-labelled probe was added. Prior to hybridization, probes were first denatured by incubation at 95 °C for 5 min, then placed onto ice. The probes were then diluted in DIG-Easy hyb at a rate of 3.5 mL of diluted probe per 100 cm2 of membrane, added to the membrane, and incubated overnight at 42 °C with rotation.To prepare dot blots, 3 µL of generic PCR product from each sample was added to 27 µL of denaturation solution  conjugate with an AP conjugate color substrate  following the DIG-detection kit, as per the manufacturer’s instructions. The color substrate was left to develop for at least 30 min. The reaction was then stopped by washing membranes in distilled water.Field surveys of Queensland garlic crops from four properties  were comA pot trial was done to investigate the distribution of individual virus species in planting propagules  from infected bulbs. Bulbs were selected from symptomatic plants of cultivar Glenlarge from a single Queensland property in 2013 and separated based on weight into large (>80 g) and small (20–30 g) bulbs. Cloves from all test bulbs were planted individually and grown in a temperature-controlled glasshouse environment. To generate approximately 100 cloves per size class for evaluation, 14 bulbs of the small size were used, whereas only seven of the larger bulbs were required. The quality of the planting material was evaluated for viability, average clove weight, and number of cloves per bulb. A total of 92 cloves produced plantlets from small sized bulbs, and 110 from the larger bulbs. The subsequent plantlets were maintained insect- and mite-free to prevent cross-infection of viruses, rated for virus symptoms and indexed for individual virus species using RT-PCR dot blot hybridization described above. Principal component analysis (PCA) in the statistical software package Minitab was employed to measure correlation between the weight (grams) of large- and small-sized bulbs and their virus infection status and average % cloves infected with individual viruses within the bulb.Direct sequencing of the coat protein (CP) coding region was completed for several representative survey samples for the various garlic viruses identified. Using Geneious software, primers were designed to amplify the partial or complete CP-encoding region of the identified viruses. The CP sequencing involved PCR amplification of 2 µL cDNA (described above) with 10 pmol targeted primers in a 25 µL reaction containing 0.3 µL Taq polymerase . PCR cycling conditions were as follows: initial denaturation 95 °C for 1 min, followed by 35 cycles of 95 °C for 15 s, 48–62 °C for 20 s, 72 °C for 60 s, and a final extension of 72 °C for 5 min. See Using the Geneious software, a global multiple sequence alignment was created, and phylogenetic trees were generated using genetic distance model Tamura-Nei and the UPGMA tree build method setting. Distances were obtained from pairwise alignments of all sequences. A bootstrap resampling method with 100 replicates was applied to generate a consensus tree with a support threshold of 50%.


The emergence or re-emergence of viruses with epidemic and/or pandemic potential, such as Ebola, Zika, Middle East Respiratory Syndrome (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus 1 and 2 (SARS and SARS-CoV-2) viruses, or new strains of influenza represents significant human health threats due to the absence of available treatments. Vaccines represent a key answer to control these viruses. However, in the case of a public health emergency, vaccine development, safety, and partial efficacy concerns may hinder their prompt deployment. Thus, developing broad-spectrum antiviral molecules for a fast response is essential to face an outbreak crisis as well as for bioweapon countermeasures. So far, broad-spectrum antivirals include two main categories: the family of drugs targeting the host-cell machinery essential for virus infection and replication, and the family of drugs directly targeting viruses. Among the molecules directly targeting viruses, nucleoside analogues form an essential class of broad-spectrum antiviral drugs. In this review, we will discuss the interest for broad-spectrum antiviral strategies and their limitations, with an emphasis on virus-targeted, broad-spectrum, antiviral nucleoside analogues and their mechanisms of action. Antiviral drug development has long focused on virus-specific approaches: the strategy to study a virus and identify a specific viral protein as a drug target in order to limit potential toxicity and to increase drug efficacy . Virus-sDrug re-purposing is advantageous in that the initial part of the drug discovery process can be bypassed including medicinal chemistry research and lead optimization as well as the safety evaluation in phase 1 clinical trials, therefore reducing considerably the research and development time. Yet, re-purposing antiviral drugs successfully is inherently dependent on the ability of an antiviral drug to be active against multiple viruses, hence, the need of a broad-spectrum antiviral effect. Historically, broad-spectrum antivirals have first been discovered by serendipity through simple screening assays against different viruses rather than through a deliberate strategy to design drugs to display a broad-spectrum antiviral effect ,18 for rHost-targeted antiviral strategies, reviewed here ,22, featA host-targeted approach often offers a higher barrier to the appearance of viral drug resistance. In practice, developing both host-targeted and virus-targeted broad-spectrum antivirals is crucial and complementary to answer the viral outbreaks to come. Virus-targeted- broad-spectrum antiviral strategies present their own challenges; the diversity of viral protein structures and sequences make the design of broadly acting compounds particularly difficult, and often result in a limited spectrum of antiviral applications. Up until now, only one class of compounds, nucleoside analogues, has shown promise for broad-spectrum antiviral applications in the clinic. Viruses adopt different replication strategies, yet all replicate their genetic material via a DNA or RNA polymerase conferring potential susceptibility to molecules resembling the natural nucleoside building blocks of their genomes; the antiviral nucleoside analogues. Many viruses also share the ability to adapt rapidly to new stress conditions which makes them vulnerable to antiviral strategies targeting their mutation frequency.Antiviral nucleoside research began in the 1960–1970s ,23 but dAlthough antiviral nucleoside analogues form a major class of antiviral drugs , the devThe definition of “broad-spectrum antiviral” can be considered a matter of scale, with some defining the term to encompass all viruses, or perhaps all RNA viruses, or some defining more narrowly to include all viruses of a particular family. The number of direct-acting and virus-targeted nucleoside analogues displaying an antiviral effect across different virus families is limited. Three nucleoside analogues stand out as broad-spectrum RNA virus inhibitors: Remdesivir, Ribavirin and T-705/Favipiravir. We will further discuss these three nucleoside analogues, their antiviral activities, and mechanisms of action.C-nucleoside analogue and a phosphoramidate prodrug that resembles adenosine monophosphate. The C-nucleoside nature of Remdesivir, with the replacement of the natural carbon–nitrogen bond between the base and the sugar, by a carbon–carbon bond, is advantageous in terms of chemical and enzymatic stability  to clearly understand how a drug is working against a specific virus. Innovation at multiple levels are required to increase discovery of broad-spectrum antiviral drugs, such as the discovery of novel biological mechanisms shared by multiple viruses and the design of medicinal chemistry approaches to target these mechanisms. Additionally, even the most active broad-spectrum antiviral drugs display a range of antiviral activities depending on the virus. Therefore, studying drug combinations to limit drug resistance development and to increase antiviral efficacy is also needed.


Few studies have used real-world data (RWD) to evaluate uUTI-associated healthcare resource use (HRU) and costs. We examined HRU and direct costs associated with appropriate This retrospective cohort study used RWD from IBM MarketScan  to examine uUTI-related HRU and costs  per index uUTI episode and during 1-year follow-up among females (age ≥ 12 years) diagnosed with uUTI from July 1, 2013–December 31, 2017 (index date). Patients had an oral AB prescription ± 5 days of the index date, and continuous health plan enrollment ≥ 6 months pre/1 year post-index date; those with complicated UTI were excluded. Patients were stratified by AB prescription as follows: AP&OP = guideline-compliant and correct duration; IA/SO = guideline non-compliant/incorrect duration or re-prescription/switch within 28 days.The study included 557,669 patients. In the commercial population , fewer patients were prescribed AP&OP (11.8%) than IA/SO (88.2%) ABs, a trend also seen in the Medicare population . In both populations, adjusted average numbers of uUTI-related ambulatory visits and pharmacy claims were lower for the AP&OP cohort than the IA/SO cohort during index episode and 1-year followup . In the commercial population, total adjusted uUTI-related costs were &194 (AP&OP) versus &274 ; in the Medicare population, total adjusted uUTI-related costs were &253 (AP&OP) versus &355  (Table 2).Table 1. uUTI-related HRU for commercial and Medicare populations calculated using the GLM modelTable 2. uUTI-related costs for commercial and Medicare populations calculated using the GLM modelOverall uUTI-related HRU and costs in the US were low during index episodes and follow-up. However, females with uUTI prescribed IA/SO ABs were more likely to incur higher HRU and costs than those prescribed AP&OP ABs, suggesting an unmet need for training to optimize uUTI prescribing per US guidelines.Madison T. Preib, MPH, STATinMED Research  Fanny S. Mitrani-Gold, MPH, GlaxoSmithKline plc.  Xiaoxi Sun, MA, STATinMED Research  Christopher Adams, MPH, STATinMED Research  Ashish V. Joshi, PhD, GlaxoSmithKline plc.


Multiple respiratory viruses can concurrently or sequentially infect the respiratory tract and lead to virus‒virus interactions. Infection by a first virus could enhance or reduce infection and replication of a second virus, resulting in positive (additive or synergistic) or negative (antagonistic) interaction. The concept of viral interference has been demonstrated at the cellular, host, and population levels. The mechanisms involved in viral interference have been evaluated in differentiated airway epithelial cells and in animal models susceptible to the respiratory viruses of interest. A likely mechanism is the interferon response that could confer a temporary nonspecific immunity to the host. During the coronavirus disease pandemic, nonpharmacologic interventions have prevented the circulation of most respiratory viruses. Once the sanitary restrictions are lifted, circulation of seasonal respiratory viruses is expected to resume and will offer the opportunity to study their interactions, notably with severe acute respiratory syndrome coronavirus 2. Several respiratory viruses can circulate during the same period and can concurrently or sequentially infect the respiratory tract, leading to virus‒virus interactions. At the host level, the course of infection of 1 virus might be influenced by prior or concurrent infection by another virus. Infection by a first virus could enhance or reduce infection and replication of a second virus, resulting in positive (additive or synergistic) or negative (antagonistic) interaction.Positive virus‒virus interaction corresponds to a co-infection that might result in an increased disease severity and pathogenesis  . ThiInduction of ISGs by a first virus might limit infection and replication of a second virus, especially if they show a differential ability to induce an IFN response or different degrees of susceptibility to immune mediators. To evade the immune system, respiratory viruses have developed a series of mechanisms that counteract the induction and antiviral action of IFNs, which might influence the type of virus‒virus interactions. Influenza viruses and SARS-CoV-2 have developed a broader range of multifaceted strategies to escape IFN induction and signaling than RSV, human metapneumovirus (HMPV) and human rhinovirus (HRV) .At the cellular level, blocking or reduction of cell surface receptors and competition for cellular resources and factors were suggested as mechanisms of negative virus‒virus interaction. For instance, the expression of neuraminidase in 293T cells infected with influenza A(H1N1) or A(H3N2) viruses can prevent a subsequent infection with retroviruses pseudotyped with a range of hemagglutinin molecules or a second IAV by removing sialic acid from the cell surface  virus reemerged during 1977 and cocirculated with seasonal influenza A(H3N2) before being replaced by the influenza A(H1N1)pdm09 pandemic virus. During the 1977–78 winter season in Japan, the percentage of children infected with H1N1 virus was lower for those infected shortly before with H3N2 virus than for persons who were not infected with H3N2 virus  in 3 schools in which sequential outbreaks were observed (During the 2009–2011 influenza A(H1N1)pdm09 virus pandemic, several countries recorded distinct influenza epidemic peaks. During 2009, only pH1N1 virus circulated during the influenza season (weeks 23–36) in most temperate countries of the southern hemisphere. In contrast, a typical seasonal H3N2 peak (week 33) preceded the first pH1N1 wave (weeks 34–38) in South Africa  has been evaluated in a ferret model . For thi,–,<7 days as assessed by viral shedding in nasal wash specimens . A fRSV and HMPV co-circulate during winter and spring and can be co-detected in nasopharyngeal swab specimens of patients. Nevertheless, the type of interaction between these 2 pneumoviruses is controversial. A study reported that the likelihood of co-detecting RSV and HMPV in patients was reduced compared with expected values , suggesting that a viral interference could occur , but the–,–Many studies reported that the 2009 autumn epidemic of HRV might have delayed the circulation of pH1N1 in several countries in Europe –, suggestDetection of RSV was associated with a reduced probability of co-detecting HRV in clinical specimens , indicating a negative virus‒virus interaction  (25). ThIn contrast to most respiratory viruses, HRV continued to circulate despite the mitigation measures put in place during the COVID-19 pandemic. HRV is a nonenveloped virus that is more resistant to hydroalcoholic disinfectant , but theSARS-CoV-2 was shown to trigger a broader up-regulation of ISGs, cytokines and chemokines in the human nasal mucosa than pH1N1 virus  are produced during replication of RNA viruses and are believed to play a role in adaptation, viral escape, and persistence , prevented disease caused by a second infection with a heterologous IBV . Factors that could predict an interference between respiratory viruses include the capacity of the interfering virus to induce a rapid IFN response; the degree of susceptibility of the second virus to immune mediators; the extent to which the different viruses counteract the induction and antiviral effects of IFN; and the differential innate immune response patterns triggered by each viruses in the upper and lower respiratory tracts.The duration of the refractory period at the host level has not been determined, but might correspond to the period of virus shedding and the associated transient innate immune response. Mathematical models that simulate the co-circulation of seasonal IAV and HRV confirmed that the temporary immunity provided by an IFN response might be sufficient to produce the asynchronous epidemic peaks recorded for these 2 viruses (The reappearance of H1N1 virus during 1977 and the 2009–2011 pH1N1 pandemic offered the opportunity to study the epidemiologic interactions between the newly circulating virus and seasonal respiratory viruses in northern and southern hemispheres and thus strengthened the concept of viral interference. During the COVID-19 pandemic, nonpharmacologic interventions have prevented the circulation of most respiratory viruses. Therefore, their potential interactions with SARS-CoV-2 could not be determined in epidemiologic studies, except in some reports at the onset of the pandemic. A systematic review and meta-analysis showed that the most common respiratory viruses co-detected with SARS-CoV-2 were influenza viruses, RSV, and HRV (Mathematical modeling predicting the timing and magnitude of epidemics caused by SARS-CoV-2 and seasonal respiratory viruses might improve public health interventions to protect persons at risk for co-infection through introduction of nonpharmacologic measures, adjustment of vaccine schedules, or use of prophylactic agents. Finally, the interfering and immunostimulatory activities of DVGs make them attractive candidates for development of prophylactic broad-spectrum antiviral drugs or vaccine adjuvant, which would be based on the concept of viral interference (


Anatidae. In this study, susceptibility of common Anatidae to a clade 2.3.4.4e H5N6 HPAI virus was assessed to evaluate their potential to be a source of infection for other birds. Eurasian wigeons (Mareca penelope), mallards (Anas platyrhynchos), and Northern pintails (Anas acuta) were intranasally inoculated with 106, 104, or 102 50% egg infectious dose (EID50) of clade 2.3.4.4e A/teal/Tottori/1/2016 (H5N6).There were large outbreaks of high pathogenicity avian influenza (HPAI) caused by clade 2.3.4.4e H5N6 viruses in the winter of 2016–2017 in Japan, which caused large numbers of deaths among several endangered bird species including cranes, raptors, and birds in Family 4 EID50 of virus seroconverted within 10 days post-inoculation (dpi). Virus was mainly shed via the oral route for a maximum of 10 days, followed by cloacal route in late phase of infection. Virus remained in the pancreas of some ducks at 10 dpi. Viremia was observed in some ducks euthanized at 3 dpi, and ≤ 106.3 EID50 of virus was recovered from systemic tissues and swab samples including eyeballs and conjunctival swabs.All birds survived for 10 days without showing any clinical signs of infection. Most ducks inoculated with ≥ 10These results indicate that the subject duck species have a potential to be a source of infection of clade 2.3.4.4e HPAI virus to the environment and other birds sharing their habitats. Captive ducks should be reared under isolated or separated circumstances during the HPAI epidemic season to prevent infection and further viral dissemination.The online version contains supplementary material available at 10.1186/s12917-022-03222-7. To date, H5 subtype high pathogenicity avian influenza viruses (HPAIVs) have spread to domestic poultry and wild birds in over 70 countries in Asia, Europe, the Middle East, and Africa since late 1996 when the virus, recognized as the precursor of present circulating viruses (Goose/Guangdong [Gs/GD]-like viruses) was isolated from a goose in Guangdong Province, China . SubsequGrus monachal) and white-naped cranes (Grus vipio), which are classified as Vulnerable according to the International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species [Branta hutchinsii leucopareia, a Critically Endangered category in the IUCN Red List) in Higashiyama Zoo and Botanical Gardens in Aichi Prefecture also died of infection [Anatidae bird species including Eurasian wigeons (Mareca penelope) and Northern pintails (Anas acuta) sharing their habitat with cranes in the Izumi Plain, and mallards (Anas platyrhynchos) and Eurasian wigeons co-housed with the cackling geese in Higashiyama Zoo. These incidents suggest that the risk of HPAIV infections in endangered bird species is correlated with the viral susceptibility of Anatidae.In the winter of 2016–2017, clade 2.3.4.4e  H5N6 HPAIVs caused many outbreaks of HPAI in poultry and wild birds in the East Asian countries –10. Japa Species . Reared nfection . Viral iAnatidae such as the American black duck (Anas rubripes), Baikal teal (Anas formosa), common teal (Anas crecca), Eurasian wigeon, lesser scaup (Aythya affinis), mallard, Mandarin duck , pintail, ruddy duck (Oxyura jamaicensis), and surf scoter (Melanitta perspicillata) [Anatidae, and that the viruses were mainly shed via the oral route. Little is known about the pathogenicity of clade 2.3.4.4e HPAIVs in Anatidae. Mandarin ducks inoculated with the clade 2.3.4.4e HPAIVs showed inapparent infection with virus shedding via the oral route [Several groups have examined pathogenicity of clade 2.3.4.4c (Group A) H5N2 or H5N8 HPAIVs, which circulated worldwide in 2014–2015, in cillata) . Most stal route ; Wang etal route  reportedAnatidae  to a clade 2.3.4.4e H5N6 HPAIV to assess their importance as a source of infection to environment and other birds, including threatened species, and to determine why clade 2.3.4.4e HPAIVs caused HPAI across East Asian countries. Migratory flyways of these ducks in East Asia were tracked using global positioning system technology in addition to classical bird banding studies [In the present study, we assessed susceptibilities of common  studies –29, and Eight of 30 captured ducks  were seropositive for influenza A virus by competitive enzyme-linked immunosorbent assay (cELISA)  of the clade 2.3.4.4e HPAIV A/teal/Tottori/1/2016 (H5N6) (Tottori/1) and observed for 10 days  seroconverted against the challenge virus by 10 days post inoculation (dpi). Among them, some ducks, including one cELISA-positive mallard (M115), shed viruses via oral route for a maximum of 10 days, peaked at 3–5 dpi. One each of wigeon and mallard, W106 and M116, respectively, also shed a relatively low titer of virus via the cloacal route. Virus was additionally recovered from a conjunctival swab of one mallard, M116, at 10 dpi, despite no virus shedding being detected in oral/cloacal samples at 7 dpi. Among the ducks inoculated with 104 EID50 of the virus, one juvenile wigeon and one juvenile mallard, W108 and M117, respectively, seroconverted (128–256 HI titers) and shed virus via the oral and/or cloacal routes. Viral shedding and antibody responses were not observed in an adult wigeon and an adult mallard  which had a pre-HI antibody titer of 2, or two Northern pintails (P127 and P128). None of the ducks inoculated with 102 EID50 of the virus shed any virus. Among them, one wigeon, W109, showed 4-fold change in its serum HI titer after the virus challenge. Notably, throughout the experiment, relatively low titers of viruses remained in the pancreas of several ducks at 10 dpi. No histopathological lesions or antigen-positive cells were observed in the tissues of ducks euthanized at 10 dpi (data not shown).Each of seven Eurasian wigeons, mallards, and Northern pintails was inoculated with 106 EID50 of the virus  of a wigeon and a pintail  and viremia was confirmed. Systemic infection was also observed in one pintail (P123) with viruses disseminated to various tissues at titers of ≤ 106.5 EID50. Notably, the three ducks with systemic infection shed relatively higher titers of the virus (104.5–5.7 EID50) via conjunctival route, accompanied by viral replication in their eyeballs. Three mallards (M111–M113) and one Northern pintail (P122) had relatively low titers of the virus in the limited tissues. No virus was isolated from two Eurasian wigeons (W101 and W103). None of the ducks had any obvious histopathological lesions at 3 dpi, other than the eyes of two pintails (P121 and P123), which showed mild nonsuppurative conjunctivitis which was composed of subconjuncitival inflammatory infiltrate and epithelial degeneration  had low titers of serum HI antibody against the challenge virus before the infection experiments, indicating that they were infected with antigenically similar influenza viruses and subsequently survived in the field. Other reports also showed that H5 HA specific antibody was detected in serum and egg yolk of wild duck populations in Asian countries [Anatidae did not shed the virus after the challenge of the clade 2.3.4.4e H5N6 HPAIV. As observed in a previous study: mallards and Mandarin ducks with homologous immunity did not contribute to virus transmission [Anatidae.In this study, three adult ountries , 31. Thesmission , 33. Antsmission . These rsmission . The ressmission . The chaAnatidae inoculated with ≥ 104 EID50 of Tottori/1 showed subclinical infection. Similar results have been reported in Mandarin ducks with clade 2.3.4.4e H5N6 HPAIVs [Pavo cristatus/Jiangxi/JA1/2016 (H5N6) [Anas crecca/shanghai/SH1/2016 (H5N6), caused no signs of infection. These results suggest that pathogenicity of clade 2.3.4.4e HPAIVs in Anatidae varies depending on the strain. Clade 2.3.4.4e H5N6 HPAIVs have multiple gene constellations (C1–C8 and more) [Anatidae flocks. The ages of the duck subjects should also be considered to explain the differences in the pathogenesis of clade 2.3.4.4e H5N6 HPAIVs. Previous reports revealed that the past H5N1 HPAIVs showed higher pathogenicity in younger domestic ducks [Anatidae captured in this study were presumed to be several months old and to have been born during the summer in Northern nesting areas such as Siberia and Alaska, and then migrated to Japan for wintering. The pathogenicity of Tottori/1 in younger ducks warrants further study.Most of the 6 HPAIVs , 33. In 6 (H5N6) , while tnd more) , 36, 37.nd more) . The relic ducks , 39. JuvAnatidae was mainly observed via the oral route as in previous experimental infection studies of Anatidae  and clade 2.3.4.4b and 2.3.4.4c H5 HPAIVs [4 EID50, in 1–5 dpi, suggesting that these duck species might be a source of infection for other wild bird species in field settings. Further, two Eurasian wigeons shed virus into the cloaca in late phase of infection. A similar observation was made in Mandarin ducks infected with a clade 2.3.4.4c H5N8 HPAIV [Anatidae, including two Northern pintails inoculated with 102 EID50 of the virus, had low titers of virus in the pancreas at 10 dpi. Such tissue tropism was also observed in Baikal teals that died of natural infection caused by a clade 2.3.4.4c H5N8 HPAIV [Anatidae. Pancreatic enzyme activity may contribute to cleavage activation of viral HA and isolation efficiency in experimental studies. In this study, virus detection in the pancreas and cloacal swabs was not correlated, indicating that the remaining viruses in the pancreas were not involved in virus shedding via the digestive tract.Virus shedding from the infected 5 HPAIVs , 20. SimN8 HPAIV . One posN8 HPAIV . It remaAnatidae. Their clinical outcomes after 3 dpi were unclear. Generally, such Anatidae are likely to be weakened and/or dead in field settings where birds have to feed by themselves, and they then become a source of infection by consumption by scavengers. Virus at titers of ≥ 104.5 EID50 was also recovered from the eyeballs and conjunctival swabs of three ducks, and mild conjunctivitis were confirmed in the Northern pintails. Continuous virus detection in conjunctival swabs has been reported in domestic ducks inoculated with H5N1 HPAIVs [Viremia and systemic infection were confirmed in two 1 HPAIVs , and ant1 HPAIVs . Scaveng1 HPAIVs ; HPAIVs 4 EID50 of the clade 2.3.4.4e HPAIV caused inapparent infection in Anatidae, sometimes accompanied by viral shedding. The number of examined Anatidae was limited and their profiles  were not uniform. However, the results reflect the circumstances of HPAIV infection in Anatidae populations in field settings. Age, history of infection, and individual differences may affect the clinical outcome. Transboundary dissemination of the virus by migratory birds, including Anatidae, is still a concern. Recently, clade 2.3.4.4b HPAIVs have become predominant in East Asian countries [Anatidae should continue to be used to estimate the risk of infection in poultry and endangered species. The results of this study contribute to the accumulation of knowledge about the susceptibility of migratory ducks to H5 HPAIVs.This study showed that ≥ 10ountries , and antountries . AssessiClade 2.3.4.4e HPAIV Tottori/1 was used for experimental infection of ducks. The strain was isolated from a fecal sample of teal in Tottori City located in the Midwestern region of Japan . The virAnatidae species  were captured at Togo and Koyama Ponds in Tottori Prefecture in Japan in two consecutive winter seasons, January and December 2019  and sex were also taken into consideration for grouping to reduce biases.Ten each of three 6, 104, or 102 EID50, then observed for clinical signs at 24-h intervals for 10 days . At the end of the 10-day period, the ducks were also checked for specific antibodies against the challenge virus in serum by HI testing. The surviving birds were euthanized by isoflurane  inhalation at an overdose after collection of conjunctival swabs and blood at 10 dpi, and their tissues  were sampled for virus isolation and histopathological study, as described below. The remaining three of each duck species were intranasally inoculated with 200 μL of allantoic fluid containing the virus at 106 EID50, then euthanized at 3 dpi. The samples were collected in the same manner as above.Seven of each duck were intranasally inoculated with 200 μL of allantoic fluid containing the Tottori/1 at 1050 was calculated using the Reed and Müench method [Portions of the tissue samples were homogenized using a Multi-Bead Shocker  at 3,000 rpm for 30 s to create a 10% (weight/volume) organ emulsion in nutrient broth medium with antibiotics. Samples serially tenfold diluted in phosphate buffered saline with streptomycin sulfate and penicillin G were inoculated into 10-day-old chicken embryos. Eggs were incubated at 35°C for 48 h. Hemagglutination testing [h method . The samAdditional file 1. Profiles and virological/serological assessments of the captured ducks.Additional file 2. Experimental designs.


Little is known on the economic implications of multi-dose 13 valent pneumococcal conjugate vaccine (PCV13) introduction in expanded program on immunization (EPI). Based on evidence of PCV13’s reduced pressure on vaccine cold chain, Benin, a third world country in West Africa, introduced the multi-dose PCV13 starting in April 2018 in its EPI program in replacement of the single-dose presentation. The objective of this study was to conduct a rapid assessment of the costs and economic impact of switching from single- to multi-dose PCV13 vial in Benin.The data collected retrospectively between January 1 and February 16, 2019 using a quantitative questionnaire was analyzed using Excel 2010 and Stata 13. Resources consumed from April 1st to September 30th, 2017 for the single-dose PCV13 and from April 1st to September 30th, 2018 for multi-dose were analyzed. For both presentations, costs analyzed included vaccines, injections supplies, waste management, cold chain, personnel , supervision and monitoring, training, social mobilization and overheads. Moreover, additional costs incurred for the introduction of multi-dose PCV13 were also collected. Costs were estimated for each presentation of PCV13 vaccine by calculating the half-year value of recurrent and capital costs, discounted at a rate of 3% for capital items. To enable comparisons, costs pertaining to 2017 were converted to 2018 equivalent values taking inflation in US$ into account.The economic costs of the single-dose PCV13 exceeded that of the multi-dose: US$ 3,708,795 versus US$ 3,698,795, respectively. Three cost items, including costs of vaccines, injection supplies, and cold chain appeared to be the main drivers of the observed reduction in costs of multi-dose PCV13. Moreover, the cost per infant vaccinated was lower with the single-dose PCV13 than the multi-dose, respectively US$ 6.28 versus US$ 10.92, and costs of vaccines wasted higher for the multi-dose PCV13.This evaluation seemed to show that the switch from single- to multi-dose PCV13 resulted in reduced economic costs of PCV13. Vaccinating more infants together with a rigorous application of vaccine open vial policy could lead to the change being more cost-effective.The online version contains supplementary material available at 10.1186/s12889-021-12108-6. Streptococcus pneumoniae is one of the leading causes of severe pneumonia in adults, and bacterial meningitis in children less than 1 year [S. pneumoniae [eumoniae . Mortalieumoniae  since 9 eumoniae , 5. Furteumoniae . In addiThe fight against pneumococcal infections includes vaccination which has been recognized as the most cost-effective strategy for reducing pneumococcal-related morbidity and mortality . PCV13 iThe introduction of a new vaccine may induce a number of changes, including a new way for communicating for social mobilization , training health personnel on how to administrate the new vaccine, expanding the chain cold, etc. . All theThis study was conducted in Benin, a third world country located in West Africa, whose population was estimated at 11.9 million in 2019 . The couBenin’s health system which has a pyramidal structure comprises three different levels . The cenHaemophilus influenzae type b, pneumococcal disease and rotavirus [push and pull approaches coexist. In the push approach, vaccines and injection supplies are distributed according to an allocation mode while in the pull scenario they are distributed according to a requisition mode. Under the push scenario, the distribution of vaccines and injection supplies are monthly supplied by a logistician using a refrigerated vehicle whereas in the pull approach health facilities obtain supplies of vaccines and injection supplies on a monthly basis from the health zone to which they belong using motorcycles or private transports. Recently, an optimized push scenario was deployed in selected health zones, with expectation that this optimized logistics system will be the only mode of vaccine distribution at peripheral levels in near future [Initiated in 1982, Benin’s EPI program fights 11 different diseases, including tuberculosis, poliomyelitis, measles, diphtheria, tetanus, pertussis, hepatitis B, otavirus . The vacr future .A retrospective survey was conducted to collect the costs of single- and multi-dose PCV13 using a structured quantitative questionnaire programmed on tablets. A purposive sampling approach which took into account all 3 levels of Bénin’s health pyramid was used to select health structures for data collection. They comprised: 1 structure at central level, 3 departments  at regional level, 17 health zones and 20 primary health centers at district level. This purposive selection of health structures was necessary to ensure availability of the needed data. The study questionnaire is provided as Additional file Agence Nationale de la Vaccination et des Soins de Santé Primaires; departmental levels with the Chief Medical Officer of the Service Départemental de la Santé Publique and of the Division Vaccination et Recouvrement des Coûts; peripheral levels with heads of technical and administrative units in charge of vaccines logistics. They aimed at collecting cold chain related costs for both presentation of PCV13’s vaccine, including vaccines, waste management, cold chain, monitoring and supervision, training, human resource, social mobilization, and overheads. These interviews were complemented by data extraction in supporting documents. In total, 16 experienced interviewers were recruited for fieldwork. They received specific training on the use of study instruments before starting the data collection.The data collection took place from January 1st to February 16th, 2019. Because the multi-dose PCV13 was introduced in April 2018, the study only had data covering a half-year. For this reason, the costs were collected over a period of 6 months, from April 1st to September 30th, 2017 for single-dose PCV13 and from April 1st to September 30, 2018 for multi-dose PCV13. The data collection encompassed various interviews at: central level with the director of the The analysis was conducted from the perspective of the government-funded health service. The economic costs of PCV13 logistics chain before/after the introduction of the multi-dose PCV13 were analyzed. Costs analyzed, including recurrent and capital costs, were identified based on standardized methods . The stuVaccine costs were estimated as the sum total of vaccine procurement and transportation. Vaccine procurement was obtained by multiplying the unit price per dose by the number of doses dispensed (doses administered plus wasted) over the studied periods for each PCV13 presentation. Unit prices per dose of single- and multi-dose PCV13 were $3.30 and $2.95, respectively . In lineCosts of syringe and safety boxes were calculated by multiplying quantities used by unit prices of each resource. Quantities used over the studied periods were obtained by summing up quantities of each resource available on April 1st to quantities received during the investigated period minus the remaining on September 30th. Similar to vaccine prices per dose, unit prices of syringes and safety boxes were searched for in UNICEF’s 2018 price list .Costs of waste management included both recurrent and capital costs incurred for the management of PCV13 wastes. The recurrent costs comprised the costs of training, per diems, maintenance of equipment, fuel for the transportation and incineration of wastes. Recurrent costs that were specific to PCV13 such as specific introductory training and per diems have been fully allocated to the vaccine. Shared costs, including maintenance, fuel, training and per diems for overall vaccination activities were appropriately allocated to each PCV13 presentation in proportion to PCV13 vaccines volume in the cold chain . CapitalCosts of cold chain, included recurrent and capital costs incurred for storage and distribution of PCV13 vaccines. The recurrent costs included the costs of storage equipment , maintenance of equipment, fuel for transportation, and per diems. Recurrent costs that were specific to PCV13 were fully allocated to the vaccine whereas shared costs were appropriately allocated to each presentation of PCV13 in proportion to the volume of PCV13 vaccines in the cold chain . The halThe costs of monitoring and supervision took only into account per diems to health personnel for the conduct of monitoring and supervision. Since they were shared costs, they have been thoroughly allocated to each presentation of PCV13 based on recommendations suggested in the literature .In addition to the costs of planning for the introduction of the multi-dose PCV13, costs of training, included refresher training for vaccines’ delivery and distribution, record keeping, data management and cold chain maintenance for both presentations of PCV13. Except the costs of training incurred for the introduction of the multi-dose PCV13 which were fully allocated to that vaccine, the costs of other training were allocated to PCV13 in proportion to the volume occupied by each presentation of PCV13 in the cold chain . HoweverSocial mobilization included the incurred recurrent and capital costs for the conduct of information, education and communication activities. Similar to costs of training, capital costs were amortized over 2 years, discounted at a rate of 3%. Because social mobilizations for the introduction of multi-dose PCV13 were specific costs, they were fully allocated to the vaccine. Social mobilization costs that were not specific have been appropriately allocated to each presentation of PCV13. However, the costs of social mobilization may only have been partial for the single-dose PCV13 since capital items were missing.Costs of personnel encompassed the opportunity costs of existing staff. Additional costs of staff recruited due to the introduction of the multi-dose PCV13 were also analyzed for the cited vaccine. The opportunity costs were obtained by multiplying the self-declared weekly working time of each health staff by the corresponding hourly wage. The latter product was multiplied by 28 weeks to obtain the half-yearly estimate of health personnel opportunity costs.Overheads included only the recurrent costs for routine functioning of the EPI program. These included stock record forms, delivery and supply forms as well as other miscellaneous expenses. Since these were shared costs, they have been appropriately allocated in proportion to the volume occupied by each presentation of PCV13 in the cold chain .The analysis was conducted in Excel 2010 and Stata 13. Monetary values were reported in US dollars, with US$1 = 559 F CFA  in 2018, and 582 F CFA in 2017 . The totThe annualized economic costs of the single-dose PCV13 were slightly higher than the multi-dose PCV13: $3,708,795 Vs $3,698,935 In spite of the reduction of the total economic costs of the multi-dose PCV13, the cost per infant vaccinated with the single-dose PCV13 was lower than that of the multi-dose: $6.28 Vs $10.92, respectively (Table Table ). The findings also showed a lower cost per vaccine dose wasted for multi-dose PCV13 compared to single-dose PCV13. However, this was not surprising since the number of vaccines wasted was higher for multi-dose PCV13.The costs of wasted multi-dose PCV13 were higher than that of the single-dose: $1,337,842 Vs $1,236,953, respectively . An annual cost evaluation may have been better since recurrent costs, depending on the level of activity may be subject changes due to seasonal variations in hospital flow. In addition, a longer time-period post-introduction would have been valuable in giving staff appropriate training on the open vial policy which is a critical component of the introduction of the multi-dose vials. Inclusion of adequate training would have increased costs and would potentially have increased the number of multi-dose PCV13 administered. This, in turn, may have contributed to reducing the cost per infant vaccinated.This evaluation seemed to show that the switch from single- to multi-dose PCV13 resulted in reduced economic costs of PCV13. This cost reduction, although modest, is globally linked to a decrease in the recurrent costs, including vaccines, injection supplies and cold chain. However, the cost per infant vaccinated was higher with multi-dose PCV13. Therefore, efforts to vaccinate more infants together with a rigorous application of vaccine open vial policy could lead to the change being more cost-effective.Additional file 1. Study questionnaire


Complete polyp resection is the main goal of endoscopic removal of large colonic polyps. Resection techniques used for their removal have evolved in recent years and endoscopic submucosal dissection (ESD), endoscopic mucosal resection (EMR) with margin ablation, cold snare polypectomy (CSP), cold snare EMR and underwater EMR have been introduced. Yet, efficacy of these techniques with regard to local recurrence rates (LRR) compared to traditional hot snare polypectomy (HSP) and standard EMR remains unclear.We aimed to analyze LRR of large colonic polyps in a systematic review and meta-analysis.MEDLINE, EMBASE, EBM Reviews, and CINAHL databases were searched to identify prospective studies reporting LRR or incomplete resection rate (IRR) after colonic polypectomy of polyps >10 mm, published between January 2011 and July 2021. Primary outcome was LRR for polyps >10 mm.6928 publications were identified, of which 34 prospective studies were included into the analysis. LRR for polyps >10 mm at follow-up (FU) up to 12 months was 11.0% . ESD  and EMR with margin ablation  significantly reduced LRR compared to standard EMR without  or with unsystematic margin ablation .Local polyp recurrence after standard EMR of large colonic polyps is high. LRR is significantly lower after ESD or EMR with routine margin ablation, so that these techniques should be considered standard for endoscopic removal of large colorectal polyps. CSP, cold snare EMR and underwater EMR should only be used within clinical trials until more high-quality data regarding LRR becomes available.None


The past 30 years have seen the growth of plant molecular farming as an approach to the production of recombinant proteins for pharmaceutical and biotechnological uses. Much of this effort has focused on producing vaccine candidates against viral diseases, including those caused by enveloped viruses. These represent a particular challenge given the difficulties associated with expressing and purifying membrane-bound proteins and achieving correct assembly. Despite this, there have been notable successes both from a biochemical and a clinical perspective, with a number of clinical trials showing great promise. This review will explore the history and current status of plant-produced vaccine candidates against enveloped viruses to date, with a particular focus on virus-like particles (VLPs), which mimic authentic virus structures but do not contain infectious genetic material. E. coli, yeast, insect cells, mammalian cells and plants. This review will focus on efforts to produce vaccine candidates against enveloped viruses in plants, with a particular focus on VLPs.As a consequence of their size and geometry, self-assembling macromolecules, such as virus-like particles (VLPs), have proven to be one of the most effective ways of eliciting an immune response in recipients ,2,3. ThuPlants have the theoretical advantages that high yields and low costs are potentially possible, they are attractive for the development of vaccines that could be administered orally, and they are very unlikely to be contaminated with an adventitious mammalian pathogen. There has therefore been considerable interest in expressing VLPs in plants as a means of producing vaccines for veterinary and medical use. Considerable success has been achieved in displaying antigens or producing VLPs from non-enveloped viruses ,5, incluThere have been essentially two approaches to the creation of VLPs of enveloped viruses in plants. The first involves identifying the virus-derived proteins that are known to self-assemble inside infected cells and expressing those proteins either through stable transgenesis or transiently. If the envelope proteins are expressed, the production of VLPs relies on the ability of these proteins to interact productively with plant membranes. Fortunately, this has frequently been found to be the case ,10,11.The alternative approach is to use a preformed scaffold to display antigenic sequences from enveloped viruses to create virus-specific VLPs . PromineHepadnaviridae, reverse transcribing virus with partially double-stranded circular DNA). During HBV infection, this protein interacts with phospholipids from the host cell membranes to form both viral and subviral particles  , and VLPs thereof, came after the initial successes with HBsAg. Antigen display of one of the major surface glycoproteins, haemagglutinin : recent work carried out in South Africa led to the production of rift valley fever virus (RVFV) VLPs in N. benthamiana via transient expression [Arabidopsis and the proteins were shown to be immunogenic when the plants were fed to mice [The success of influenza VLP production in plants has had a knock-on effect on the production of VLPs of the pression . Notablypression . Previoupression . Similar to mice .Coronaviridae), has mobilised the scientific community, and the plant molecular farming community is no exception [Clostridium thermocellum b-1,3-1,4-glucanase which has previously been used with influenza HA [The ongoing COVID-19 pandemic, caused by SARS-CoV-2  to produce a VLP vaccine candidate against the new coronavirus threat. This VLP consists of the SARS-CoV-2 S protein with a number of modifications: a few stabilising point mutations, a plant signal peptide instead of the native sequence, and the transmembrane domain and cytoplasmic tail of S were replaced with the equivalent sequences from influenza H5 HA , a stratN. benthamiana and demonstrated basic immunogenicity. There were also numerous examples with veterinary coronaviruses, such as the production of stable transgenic lines of Arabidopsis [N. benthamiana [Nicotiana tabacum [N. benthamiana and was shown to stimulate neutralising antibody production in mice [Before the SARS-CoV-2 pandemic, there were several attempts at producing vaccine candidates against several coronaviruses in plants. Li et al.  expressebidopsis  and potabidopsis  expressibidopsis ,77 or fubidopsis . Later wthamiana  or trans tabacum . More re in mice . Moreove in mice .N. benthamiana. Preliminary findings suggest the formation of VLPs , research has shown that several crop species can generate sufficient Rhabdovirus antigens in a reasonable biomass to make introduction of the vaccine via feeding viable without significant processing, with reports of 1% total soluble protein, or 20–35 µg G-protein per gram fresh weight. These edible vaccine candidates focused on crops amenable to genetic transformation: spinach [Alongside attaining reasonable yields in the commonly used tobacco species  could induce neutralizing antibodies in trout [N. benthamiana [Although much of the focus for plant-derived rabies vaccines has centred on terrestrial infection, Rhabdoviruses are also an important infectious agent in marine environments . With aqus VHSV, , althougin trout , indicatthamiana , though N. benthamiana followed by plant cell wall lysis as the basis for VLP extraction [Despite the fact that this virus family comprises both animal- and plant-infecting viruses, there are no published examples to date of plant-produced enveloped rhabdovirus VLPs. There is, however, a patent that describes a method of doing just this for rabies VLPs, involving the transient co-expression of M and G proteins in traction . PerhapsFlaviviridae (non-segmented positive-strand RNA viruses) in plants have focused on hepatitis C virus (HCV), with limited success. There are numerous examples of expression of an immunogenic epitope from the E2 surface glycoprotein , notably the E surface glycoprotein in transgenic rice, which was shown to elicit an immune response after oral or intraperitoneal administration into mice . The domormation .N. benthamiana [N. tabacum [N. benthamiana, and that dEDIII could be displayed on the surface of HBcAg core-like particles, a strategy which was later confirmed by Pang et al. [N. benthamiana results in the formation of prM-E VLPs, which are immunogenic in mice [The RIC technology mentioned above had previously been used to demonstrate immunogenicity in mice of a plant-produced vaccine candidate against dengue virus (DENV), through the fusion of domain III of the E protein of DENV (dEDIII) to a chimeric antibody construct . This isthamiana  and stab tabacum . Martíne tabacum  showed tg et al.  to produg et al.  and lettg et al.  has alsog et al. . Finally in mice . InteresTogaviridae) vaccines arose after an outbreak of chikungunya virus (CHIKV) in the Americas in 2013–2014 [Interest in using plants as a cost-effective production platform for Alphavirus  was efficiently produced and self-assembled into capsid-like particles when expressed alone or as part of ORF2, indicating that autoproteolytic cleavage of C occurs in plants B,C. HoweRetroviridae) are too numerous to review exhaustively here, and readers are referred to previous reviews [The examples of plant-produced vaccine candidates against human immunodeficiency virus , which causes significant disease burden in both farmed poultry and wild bird populations [N. benthamiana [N. tabacum [This review would be incomplete without a mention of Newcastle disease virus  surface glycoprotein (GP) was expressed in transgenic tobacco [N. benthamiana as part of an immune complex strategy (similar to what has been described above for flaviviruses) and was found to be protective in a mouse lethal challenge model [Pneumoviridae) by fusing small epitopes of the G protein to alfalfa mosaic virus coat protein [Paramyxoviridae) have been expressed in plants: a polyepitope sequence was expressed in transgenic carrot [This review has focused on the virus families for which there has been the most progress in terms of making antigens through plant molecular farming methods. There are, however, examples from other families of enveloped viruses which deserve mention. For example, a synthetic multiepitope peptide from ebolavirus ( tobacco , and thege model ,175. The protein ,177. Furc carrot , and fulc carrot ,180, carc carrot , and letc carrot , all of c carrot .The past 30 years have seen the development of plant-produced vaccine candidates against enveloped viruses of both medical and veterinary concern. The main challenges have included low expression of antigen, concerns about plant glycosylation patterns, and difficulties with extraction and purification. The first issue has been addressed through a combination of improvements in plant transient expression systems ,185 and While examples of plant-produced enveloped virus vaccine candidates can be found from a wide variety of enveloped virus families, the only examples of products clinically tested in humans to date are hepatitis B virus ,20, infl