Caenorhabditis elegans infection by Orsay virus is dependent upon lipids and that mutation of the master regulator of lipid biosynthesis, sbp-1, reduced Orsay virus RNA levels by ~236-fold. Virus infection could be rescued by dietary supplementation with lipids downstream of fat-6/fat-7. Mutation of a zinc transporter encoded by sur-7, which suppresses the lipid defect of sbp-1, also rescued Orsay virus infection. Furthermore, reducing zinc levels by chemical chelation in the sbp-1 mutant also increased lipids and rescued Orsay virus RNA levels. Finally, increasing zinc levels by dietary supplementation led to an ~1,620-fold reduction in viral RNA. These findings provide insights into the critical interactions between zinc and host lipids necessary for virus infection.Viruses utilize host lipids to promote the viral life cycle, but much remains unknown as to how this is regulated. Zinc is a critical element for life, and few studies have linked zinc to lipid homeostasis. We demonstrated that IMPORTANCE Orsay virus is the only known natural virus pathogen of Caenorhabditis elegans, which shares many evolutionarily conserved pathways with humans. We leveraged the powerful genetic tractability of C. elegans to characterize a novel interaction between zinc, lipids, and virus infection. Inhibition of the Orsay virus replication in the sbp-1 mutant animals, explained by the lipid depletion, can be rescued by a genetic and pharmacological approach that reduces the zinc accumulation and rescues the lipid levels in this mutant animal. Interestingly, the human ortholog of sbp-1, srebp-1, has been reported to play a role for virus infection, and zinc has been shown to inhibit the virus replication of multiple viruses. However, the mechanism through which zinc is acting is not well understood. These results suggest that the lipid regulation mediated by zinc may play a relevant role during mammalian virus infection. Caenorhabditis elegans is an animal model with many genes and biological processes that are evolutionarily conserved in higher eukaryotes. Thus, many fundamental discoveries in this model organism have been extrapolated into humans, such as apoptosis, RNA interference (RNAi), and microRNAs  is a transcription factor that belongs to the basic helix-loop-helix leucine zipper family and is important for the homeostasis of lipids in the cell. In mammals, there are two srebp genes, srebp1 and srebp2. srebp1 is mainly involved in the expression of fatty acid biosynthesis genes whereas srebp2 is involved in cholesterol biosynthesis (srebp is located in the endoplasmic reticulum (ER) membrane as a precursor. When specific cellular lipids are low, the protein is transported to the Golgi complex and released by proteases. Then, the mature srebp is translocated into the nucleus, where it induces the transcription of more than 30 lipogenic genes  desaturases fat-6/fat-7, the stearoyl-coenzyme A desaturase fat-5, and the monomethyl branched-chain fatty acids elo-5/elo-6 ethane-1,2-diamine]. Finally, direct treatment of wild-type (WT) animals with zinc reduced Orsay virus RNA levels. In these studies, we were able to observe correlated changes in lipid abundance and Orsay virus RNA levels, demonstrating a connection between zinc, lipids, and virus infection.Here, we show C. elegans, we quantified lipid abundance in live animals by lipid staining and microscopy. We employed a previously described transcriptional reporter strain, jyls8;rde-1(ne219), which is hypersensitive to Orsay virus and carries an integrated transcriptional green fluorescent protein (GFP) reporter driven by the pals-5 promoter that is induced by Orsay virus and enables visualization of viral infection by fluorescence microscopy , daf-3(ok3610 and m3D790), daf-16(m486 and mgDF50), and mdt-15(tm2182) displayed viral RNA levels that were reduced ~16-, ~14.5-, ~11.6-, and ~23.6-fold, respectively (nhr-80(tm1011) did not display reduced viral RNA levels. Interestingly, sbp-1(ep79), which has a deletion of 2.1 kb resulting in partial loss of function (sbp-1(ep79) is playing a major role during Orsay virus infection in C. elegans.To determine whether Orsay virus depends on lipids for infection, we quantified Orsay virus RNA levels in animals depleted of lipids. First, we used RNA interference to knock down the major transcription factors,  elegans . RNAi kne, dpy-3 . To correctively . Of all function , caused function . These rsbp-1(ep79) mutant is due to SBP-1, we evaluated Orsay virus infection in a transgenic sbp-1(ep79) strain that overexpresses SBP-1 fused to GFP    . First, -1(ep79)  and D. L animals . This resbp-1 is a transcriptional activator of the expression of multiple lipogenic enzymes in three different branches  desaturases and which function redundantly to synthesize oleic acid from stearic acid, reduced the Orsay virus RNA levels; however, knockdown of elo-2, which is involved in the synthesis of stearic acid, or the pathways represented by fat-5 or elo-5 and elo-6 did not alter Orsay virus RNA levels.branches  30, 45), 45sbp-1y branch  and B. Kelo-2, which is an essential gene (fat-6(tm331);fat-7(wa36) double mutant animals, which are deficient in oleic acid production (fat-5(tm420) mutant animals, which are deficient in the production of palmitoleic acid (elo-5(gk182) and elo-6(gk233) mutant animals Orsay virus RNA levels were reduced by ~65-fold and ~10-fold, respectively  mutant, there is accumulation of the saturated fatty acid stearic acid, whereas several lipid products like oleic acid and linoleic acid, or monomethyl branched-chain fatty acids like C15iso and C17iso are reduced (sbp-1(ep79) mutant for Orsay virus infection, we biochemically supplemented the animals with a range of lipids. After 3 days postinfection (dpi), the animals were collected, and the viral RNA was quantified by quantitative reverse transcription-PCR (qRT-PCR). As expected, feeding with stearic acid had no impact on sbp-1(ep79) animals, as stearic acid is upstream of the genes regulated by sbp-1. Likewise, we did not find rescue of the viral RNA levels for animals fed with oleic acid or linoleic acid  mutation was directly affecting the viral replication step of the Orsay virus life cycle. We employed a previously described replicon system for Orsay virus based on an extrachromosomal array of plasmids of the entire Orsay virus WT RNA1 segment that encodes a WT RNA-dependent RNA viral polymerase (RdRp)  under a heat shock promoter (sbp-1(ep79) mutant to generate the replicon system in this background and compare the viral RNA levels of this strain with those of wild-type animals. The replicon system was induced by heat shock, and the Orsay virus RNA levels were quantified by qRT-PCR. In the sbp-1(ep79) mutant carrying the RNA1 WT replicon {WUM108 }, there is no statistical difference from WUM110 , and these viral RNA levels were ~55-fold lower than those of the N2;PHIP::RNA1WT animals  mutant demonstrated that lipid deficiency of the sbp-1 mutant is important. However, the factors involved in lipid homeostasis are still not clear. Interestingly, a forward genetic screen identified sur-7 as a suppressor of the sbp-1(ep79) mutant that restores lipid levels (sur-7 encodes a member of the cation diffusion facilitator family and is involved in zinc metabolism (sur-7(ku119) could rescue the lipid levels in the sbp-1(ep79) mutant, we performed LipidTox staining. We confirmed that the sur-7(ku119);sbp-1(ep79) double mutant rescued lipid levels to those of wild-type animals (sur-7(ku119);sbp-1(ep79) background compared to sbp-1(ep79) . Likewise, to investigate if the virus infection in the sbp-1(ep79) mutant could be rescued, we challenged the sur-7(ku119);sbp-1(ep79) double mutant animals with Orsay virus. Orsay virus RNA levels in the double mutant were increased compared to the sbp-1(ep79) mutant and similar to those of wild-type animals  mutant. If a high concentration of zinc is depleting the lipid levels in the sbp-1(ep79) mutant and this is reducing the Orsay virus infection, then reduction of zinc levels in the sbp-1(ep79) mutant should rescue the lipid production and rescue the virus infection. If zinc is not important for lipid homeostasis, then zinc depletion should not rescue the lipid levels and the Orsay virus infection. Addition of TPEN, a membrane-permeant zinc chelator (sbp-1(ep79) mutant up to ~2.6-fold (sbp-1(ep79) mutant to levels similar to those in wild-type animals (sbp-1(ep79) mutant play a role in the reduction of the lipid levels, thereby preventing efficient Orsay virus infection  ectopic supplementation of various lipids, (ii) mutation of the sur-7 gene, and (iii) pharmacological depletion of zinc with TPEN.Because it is known that mutation of 1 mutant , we usedchelator , 75, to 2.6-fold  and E. L animals . This reSince reducing zinc levels helps to recover the Orsay virus infection, this suggests that increasing the zinc concentration might hurt the virus. We tested this prediction and supplemented zinc into the medium of wild-type N2 animals. Supplementation of zinc in wild-type animals reduced the viral RNA levels ~1,620-fold compared to their respective controls in standard medium . TreatmeC. elegans and its conservation of many pathways with mammals make it an excellent reductionist model to explore host-virus interactions. Use of Orsay virus enables the study of host-virus interactions in vivo in a natural multicellular host. Here, we explored the role of lipids in Orsay virus infection. We demonstrate that Orsay virus impacts lipid homeostasis by reducing the amount of lipids ~60% by 48 hpi in animals, suggesting that Orsay virus alters lipid metabolism during viral infection. Interestingly, in human cells, dengue virus similarly reduces lipid abundance by 60% at 24 to 48 hpi  mutants accumulate fatty acids like palmitic and palmitoleic acid, which belong to the fat-5 branch, but have reduced levels of oleic acid, linoleic acid, and C15iso/C17iso, which correspond to the fat-6/fat-7 and elo-5/elo-6 branches, respectively (sbp-1(ep79) mutants biochemically with multiple lipids from the fat-6/fat-7 branch, including α-linoleic acid and γ-linoleic acid and dihomo-γ-linoleic acid, rescued viral RNA levels, but stearic acid, arachidonic acid, C15iso, or C17iso did not. The inability of stearic acid to rescue is predicted since stearic acid is the precursor of the fat-6/fat-7 enzymes and the sbp-1(ep79) mutant accumulates stearic acid. The observation that multiple lipids downstream of fat-6/fat-7 rescued Orsay virus RNA levels confirms the importance of this branch of lipid metabolism for Orsay virus. We did not observe rescue of the sbp-1(ep79) mutant by supplementation of C15iso or C17iso even though mutants with mutations in elo-5 and elo-6, the genes that synthesize these two lipids, respectively, have lower levels of Orsay virus infection. One possible explanation is that in the sbp-1(ep79) mutant there is a reported 50% reduction in the C15iso/C17iso lipid levels whereas in elo-5 and elo-6 mutants, which have a deletion of 379 bp and 184 bp of the first exon, respectively, the reduction is likely to be greater; therefore, the levels of these two lipids may not be limiting factors in the sbp-1 background  mutant, indicating that one or more lipids regulated by sbp-1 are needed for virus RNA replication. While a number of studies have defined the importance of srebp-1, the human ortholog of sbp-1, for viral infection, none of the studies have implicated a specific stage of the viral life cycle that is dependent on srebp1 (sbp-1(ep79) mutant, although it is possible that additional other steps of the viral life cycle may be also affected.Lipids could be required for one or more steps of the Orsay virus life cycle, such as viral entry, proper release and trafficking of infectious particles into the cytoplasm, replication of viral RNA, viral assembly, or egress. To better define the stage at which ransgene . We founn srebp1 , 37, 81.sbp-1 are important for Orsay virus infection. The exact mechanism by which these lipids are necessary for an efficient virus replication is still unknown, but several possibilities center on roles involving the viral replication center. It has been shown that oleic acid plays a role in the viral replication step of hepatitis C virus (HCV), as its depletion disrupts the integrity of membranous HCV replication centers and renders HCV RNA susceptible to nuclease-mediated degradation  mutant lipid defect found that mutation of sur-7, which encodes a transporter of zinc, restores lipid homeostasis , CE548 (SBP-1:GFP), drh-1(ok3495), sbp-1(ep79), nhr-49(ok2165 and nr2041), daf-3(ok3610 and mgDf90), daf-16(mu86 and mgDf50), mdt-15(tm2182), nhr-80(tm1011), fat-5(tm420), fat-6(tm331);fat-7(wa36), elo-5(gk182), elo-6(gk233), and sur-7(ku119) strains were obtained from the Caenorhabditis Genetics Center (CGC); these strains were maintained under standard lab culture conditions unless otherwise specified. In brief, animals were fed Escherichia coli OP50 on nematode growth medium (NGM) dishes in a 20°C incubator and moved every 3 days to a new NGM dish seeded with E. coli OP50  larvae were seeded. L1 larvae were allowed to recover for 20 h at 20°C prior to infection. Orsay virus filtrate was thawed at room temperature and then diluted 1:10 with M9 buffer. For each well, 20 μL of 2.5 × 105 tissue culture infective doses (TCID50)/mL (multiplicity of infection [MOI] of 10) of virus filtrate was added into the middle of the bacterial lawn and incubated at 20°C. Three days after infection, animals were collected into 1.5-mL Eppendorf tubes by washing each well with 1 mL of M9 buffer and then pelleted by spinning for 1 min at 376 × g in a benchtop centrifuge. M9 supernatant was removed, 300 μL TRIzol reagent (Invitrogen) was added to the tubes, and then the tubes were frozen in liquid nitrogen. For each experiment, three replicate wells were used for each infection condition unless otherwise indicated. Total RNA from infected animals was extracted using Direct-zol RNA miniprep (Zymo Research) purification according to the manufacturer’s protocol and eluted into 30 μL of RNase/DNase-free water.Orsay virus was prepared by liquid culture as described previously , filtereE. coli OP50 and dispensed on NGM agar dishes to yield a final concentration of 10×. A synchronized population of the jyls8;rde-1(ne219) strain (WUM31), in stage L1, was transferred to the dish and incubated for 20 h. Then the animals were mock infected or infected with 200 μL of a 1:10 dilution of Orsay virus in M9 buffer and collected at 48 h postinfection. Twenty-five animals were transferred to 384-well plates containing 60 μL of phosphate-buffered saline (PBS) and anesthetized with 25 mM tetramisole in M9. Total fluorescence intensity was acquired with the ArrayScan V HCS reader . Three independent experiments were performed in duplicate, making a total of 150 animals quantified per condition. Statistical testing was done by the parametric two-tailed t test or one-way analysis of variance (ANOVA) to compare two or more than two different groups, respectively.LipidTox Red Neutral lipid  was diluted in E. coli strain HT115, carrying double-stranded RNA expression cassettes for genes of interest, was induced using established conditions and then seeded into a six-well NGM dish. dpy-3, sbp-1, mdt-15, nhr-49, daf-3, daf-16, nhr-80, fat-5, fat-6, fat-7, elo-2, elo-5, and elo-6 RNAi clones were from the Ahringer RNAi library  was added for RNA extraction.RNAi feeding was used for gene knockdown as described previously . E. coli library . Twenty in vitro transcripts. Primers GW314 and GW315 and probe OrV_RNA1, which target the Orsay virus RNA1 segment, were used to quantify Orsay virus RNA1 abundance  to quantify Orsay virus RNA as previously described . Brieflybundance . To cont elegans . At leasC. elegans N2 young adults were injected by using a microinjection system (Zeiss). Sets of three microinjected animals were placed on E. coli OP50-seeded 6-well NGM dishes and maintained at 20°C. F1 animals displaying the fluorescent marker at the pharynx were individually transferred onto new E. coli OP50-seeded 6-well NGM dishes 3 to 5 days after injection. The stable transgenic array containing F2 animals was selected and maintained by picking 5 transgenic progeny animals each time onto a new E. coli OP50-seeded 6-well NGM dish  and dead viral polymerase (RNA1 DP), were microinjected into the gonad of animals to generate stable transgenic lines . BrieflyNGM dish .sbp-1(ep79) mutants and genotyped by PCR to generate the  and the  animals. sbp-1(ep79) mutant animals with the transgenic marker were selected and challenged with Orsay virus and assayed for virus replication by qRT-PCR.The transgenic RNA1 WT or dead polymerase animals were crossed by a standard method with the Six-well NGM dishes were prepared with an aqueous solution of NP-40 (Sigma) to a final concentration of 0.01% and supplemented with 1 mM concentrations of any of the following reagents: stearic acid , oleic acid , linoleic acid , α-linoleic acid , γ-linoleic acid , stearidonic acid , dihomo-γ-linoleic acid , eicosatetraenoic acid , ω-3-arachidonic acid , arachidonic acid , eicosapentaenoic acid , C15iso , C17iso , and C13iso (Chem Cruz). Dishes without lipids were also prepared. A synchronized population of 500 L1 animals was added to the wells and incubated for 36 h. Then the animals were challenged with Orsay virus for 3 days and collected for RNA extraction and qRT-PCR. Three independent experiments in triplicate were performed.4) or manganese chloride (Cl2Mn) were prepared ethane-1,2-diamine (TPEN), a zinc-specific chelator (Sigma-Aldrich) (Depletion of zinc was performed by supplementing NGM agar dishes with 1 μM Aldrich) .The imaging of animals was carried out using a Zeiss Axio Imager M2 inverted fluorescence microscope equipped with a Hamamatsu Flash4.0 complementary metal oxide semiconductor (CMOS) camera for fluorescence. Briefly, animals were collected and anesthetized with 25 mM tetramisole and then put on a 2% agarose pad with a coverslip (5 by 5 cm) on top. Images were acquired from both fluorescence channels and bright-field channels.


The significant increase in monkeypox cases that was reported at the beginning of 2022 was notable. The resurgence of viral zoonosis is especially concerning, given the current and recent COVID-19 epidemic. There are worries that a new pandemic may be beginning due to the virus that causes monkeypox spreading so quickly. This article aimed to provide an overview of the epidemiology, pathogenesis, and clinical symptoms of monkeypox. It has been known that monkeypox was primarily prevalent in Central and West Africa, but in recent years, cases of monkeypox infections have been reported around the world. The transmission of the infection to humans has been connected to exposure to a diseased animal or person's excretions and secretions. Various studies indicate that monkeypox clinically manifests as fever, fatigue, and a rash of smallpox-like lesions and can cause various complications, including pneumonia, encephalitis, and sepsis, which, when not properly managed, can lead to death. Those living in remote and forested areas, taking care of individuals infected with monkeypox, and trading and taking care of exotic animals are some of the risk factors for monkeypox. Men having sex with men are also at higher risk of contracting monkeypox. When dealing with individuals who have high-risk factors and come with new-onset progressive rashes, it is necessary for clinicians to highly suspect monkeypox. This review will serve as reference material and a supplement to the existing literature that will assist in the proper management and prevention of monkeypox. Monkeypox  is a zoonotic orthopoxvirus that incidentally causes a smallpox-like illness in people, however, with noticeably reduced mortality. This virus is clinically relevant because it is native to Western and Central Africa, and incidences in the Western world have been connected to the exotic pet trade and foreign travel. Before smallpox's eradication and the subsequent lack of vaccination campaigns, vaccination offered coincidental immunity to the Mpox virus. Nevertheless, monkeypox's rise to clinical significance was made possible by these circumstances. This article describes the prevalence and pathogenesis of monkeypox and reviews the presentation of an individual infected with the Mpox virus with the motive to assist in the diagnosis, management, and prevention of Mpox disease.The monkeypox virus, a member of the genus Orthopoxvirus, is responsible for the uncommon viral disease known as monkeypox. The Orthopoxvirus genus also comprises the smallpox-causing variola virus and the cowpox virus. The orthopoxvirus that causes monkeypox belongs to the poxviridae family. Poxviruses are brick-shaped and possess a lipoprotein-sheathed linear double-stranded deoxyribonucleic acid (DNA) genome ,2. OtherThe term "monkeypox" was changed to "Mpox" in November 2022 by the World Health Organization (WHO) . The oriPrevalence and incidenceMpox was a zoonotic illness that was most prevalent in the Democratic Republic of the Congo and was indigenous to Central and Western Africa. A study suggested African rodents are the natural reservoir, even though the disease was initially discovered in captive monkeys (thus the name). Humans, mice, squirrels, monkeys, rats, and prairie dogs have all contracted the infection . Two claHuman Mpox incidences and occasional clusters have been identified outside of Africa. Centers for Disease Control and Prevention (CDC) documented that in 2003, Gambian giant rats were smuggled from Ghana and infected nearby prairie dogs that were on sale as pets in the Midwest of the United States. A total of 53 cases of Mpox in humans were the outcome of this . An inciSeveral risk factors correlate with Mpox infection. Another study discovered that individuals who live in densely forested and rural parts of Central and Western Africa, handle and prepare bush meat, provide care to others who are sick with the Mpox virus, and who have not had the smallpox vaccine are at risk for Mpox infection. The likelihood of infection has also been linked to the male gender. Society expects men to routinely hunt and interact with wild animals, and this predisposes them to infections . A studyTransmissionUnlike the smallpox virus, the monkeypox virus also infects animals. Small mammals or monkeys seem to make up the majority of the afflicted animals . It has  [Monkeypox is primarily transmitted to humans from infected animals, especially rodents and primates. The transmission of disease to humans has been linked to exposure to diseased animal secretions and excretions ,19. The  -22. ThisThe virus can also be transmitted from one person to another through close contact with infected respiratory secretions, such as saliva or mucus, or through contact with skin lesions or other bodily fluids. Sharing drinking cups, sharing food, and sharing close quarters for sleeping were all linked to higher transmission risks. The virus appears to propagate largely through oral mucosae . WHO  into the body, it infects and replicates in cells of the immune system, particularly dendritic cells and macrophages . These cOverall, the immune system's response to the virus also plays a huge role in the course of monkeypox infection; severe or mild symptoms could be a result of the immune system's response . Also, rThe pathogenesis of the virus has also changed as a result of genetic mutations and alterations. According to meta-analysis research, children were primarily impacted by the monkeypox virus in the 1970s. The average age of those affected has risen as a result of genetic mutation, and in the year 2010, 21 was reported to be the average age of those infected. The monkeypox virus is undergoing fast alterations that could make it more dangerous and lethal .Clinical manifestationsThe initial symptoms of Mpox include fever, body aches, lymphadenopathy, and headache. The face and surrounding areas are more severely impacted than the trunk. Mouth and vaginal mucosal membranes could potentially be affected. Mouth mucosal lesions appear after one to two days, immediately followed by skin lesions on the face and extremities, including palms and soles. These lesions are centrifugally concentrated. There may or may not be any spread of the rash to the rest of the body. The lesions may range from a few to thousands in number. The lesions progress through macular, papular, vesicular, and pustular phases during the next two to four weeks. Lesions are described as hard, deeply seated, and 2 to 10 mm in size, changing synchronously. Before the formation of crusts, lesions stay in the pustular phase for five to seven days. Over the following seven to 14 days, crusts develop and desquamate, and the illness clears itself three to four weeks after the beginning of symptoms in many cases. Once all of the crusts have fallen off, the patient is no longer regarded as contagious .In comparison to other illnesses like chicken pox, the monkeypox lesions take longer to turn into crusts. Additionally, the lesions might range from one to thousands. In a relatively small number of individuals, the corneal and conjunctival mucosa were affected. Lymphadenopathy is regarded as a defining characteristic of the monkeypox virus and can be used to distinguish monkeypox from other pox illnesses ,30. The Up to 58% of the 291 people who were aware of their first symptoms available reported having at least one prodromal symptom; the illness started with a rash for 42% of individuals. The most common body parts with rash were the genitalia (46%), arms (40%), face (38%), and legs (37%); of the 718 Mpox patients who reported rash, 238 33%), 126 (18%), 98 (14%), and 256 (36%) had rash in one, two, three, or more body locations. Another study found that mouth sores, vomiting, and hospital stay longer than 48 hours were all independently correlated with these symptoms . The MSM%, 126 (1Monkeypox has been associated with various complications. In severe cases, monkeypox can lead to complications such as pneumonia, encephalitis, and sepsis. Secondary bacterial infections, such as skin infections, can arise in monkeypox patients and cause serious disease or even death. This is especially true for persons with compromised immune systems, including those with HIV/AIDS. Rarely, individuals with monkeypox may experience a severe immunological response to the virus, described as a "cytokine storm," in which the body is severely inflammatory and damaged ,35. It'sMonkeypox is a rare viral disease that occurs majorly in Central and West Africa. Over recent years, cases of Mpox infections have been reported around the world. It is transmitted to humans from infected animals and can also be transmitted from person to person. The transmission of disease to humans has been linked to exposure to a diseased animal or person's excretions and secretions. The disease is characterized by fever, fatigue, and a rash of smallpox-like lesions, and it can lead to complications such as pneumonia, encephalitis, and sepsis which may be fatal. Monkeypox's quick spread has exposed the inadequacy of precautions used to stop outbreaks. Animals being transported internationally must be quarantined and tested for the monkeypox virus. When examining patients, particularly the high-risk population, a strong index of suspicion must be in place. Mpox vaccination should be started in high-risk groups such as the immunocompromised, MSM, and people who handle animals that have traveled internationally, particularly rodents and other small mammals. To make an early diagnosis and start quick treatment, doctors need to be conscious of the early clinical manifestations of the monkeypox virus. Until all lesions have healed, patients with proven or suspected monkeypox infections must be quarantined. More study is required to develop more efficient preventive and treatment methods.


Nodaviruses are small bipartite RNA viruses and are considered animal viruses. Here, we identified two novel noda-like viruses (referred to as rice-associated noda-like virus 1 (RNLV1) and rice-associated noda-like virus 2 (RNLV2)) in field-collected rice plants showing a dwarfing phenotype through RNA-seq. RNLV1 genome consists of 3335 nt RNA1 and 1769 nt RNA2, and RNLV2 genome consists of 3279 nt RNA1 and 1525 nt RNA2. Three conserved ORFs were identified in each genome of the two novel viruses, encoding an RNA-dependent RNA polymerase, an RNA silencing suppressor, and a capsid protein, respectively. The results of sequence alignment, protein domain prediction, and evolutionary analysis indicate that these two novel viruses are clearly different from the known nodaviruses, especially the CPs. We have also determined that the B2 protein encoded by the two new noda-like viruses can suppress RNA silencing in plants. Two reverse genetic systems were constructed and used to show that RNLV1 RNA1 can replicate in plant cells and RNLV1 can replicate in insect Sf9 cells. We have also found two unusual peptidase family A21 domains in the RNLV1 CP, and RNLV1 CP can self-cleave in acidic environments. These findings provide new knowledge of novel nodaviruses. Nodaviridae . C. CNodaviaviridae . Becauseaviridae . Based oaviridae a and encaviridae . Throughaviridae d, thus, aviridae . This doaviridae . In thisaviridae . In addiaviridae , indicatRNLV2 genome is also bipartite. RNLV2 RNA1 and RNA2 are 3279 and 1525 nt in length, respectively b. RNA1 cNodaviridae. The RNLV2 CP was found to be clustered in a large clade together with the CPs of betanodaviruses and some unclassified nodaviruses infecting different hosts  a. The RNnt hosts b, even tnt hosts .Nodaviridae, due to the differences in viral protein sequences, host ranges, and phylogenetic relationships.The taxonomy of nodaviruses is decided according to the genetic diversities of their RNA2 segments . TherefoN. benthamiana plants. The result showed that in the presence of RNLV1 B2 or RNLV2 B2, strong green fluorescence was observed in the infiltrated leaf regions, similar to that observed in the leaf regions co-expressing mGFP5 and the P19 of tomato bushy stunt virus (TBSV)  a. As expsilenced a. Westersilenced b. In a sat 9 dpi c. In conat 9 dpi c. Plantsat 9 dpi d. Thus, eGFP gene before the stop codon (TAG) of the ORF1b (B2 gene) to produce a plasmid pCB301-RNLV1 RNA1-B2:eGFP (referred to as pRNLV1 RNA1wt-B2:eGFP thereafter)  a. If theng sites  and prodby 3 dpi c, indicaby 3 dpi d.v/v) mixed and then inoculated to insect Sf9 cells or rice plants. Sterile water was used as the negative control. Next, P1-supernatant was extracted from the viral RNA-transfected Sf9 cells and then used to inoculate fresh Sf9 cells or rice plants. Abnormal cellular changes including deformity and rupture were observed in the P1-supernatant-inoculated Sf9 cells by 2 dpi (data not shown). Lysate of supernatant-inoculated Sf9 cells was also prepared and examined under an electron microscope. The result showed that the lysate sample contained spherical virions of about 33 nm in diameter  CP occurs in acidic environments . Te. Te47]. Betanodavirus, due mainly to its low CP sequence homology with that of the known betanodaviruses. The members in the genus Betanodavirus share over 80% CP aa sequence homology [RNLV2 encodes a CP without an endopeptidase domain, the same as some unclassified nodaviruses that have different hosts . Althoughomology ,60, whilN. benthamiana plants expressing the MP of tobacco mosaic virus (TMV) or red clover necrotic mosaic virus (RCNMV), FHV moved systemically in the plants and accumulated in young non-inoculated leaves [N. benthamiana leaves expressed an MP from a different plant virus, such as sc4 of sonchus yellow net virus [N. benthamiana leaves through agro-infiltration. By 6 dpi, green fluorescence was found again in single cells, the same as that found in the leaves expressing PVX-GFPΔp25 alone (data not shown), indicating that PVX-GFPΔp25 could replicate in a single plant cell but could not move between plant cells. These results showed that the proteins encoded by RNLV1 or RNLV2 could not complement the cell-to-cell movement of PVX-GFPΔp25 mutant.Movement protein (MP) is a viral protein that can assist virus intercellular and systemic movement in plants. It is known that FHV can replicate in plant cells but cannot move systematically in plants . After Fd leaves . To inved leaves . It is ret virus . To testIt was reported that expression of the B2 protein of red-spotted grouper nervous necrosis virus (a betanodavirus) in fish cells induced hydrogen peroxide production and cell death ,66. BecaN. benthamiana plants. Unfortunately, all the inoculated plants remained asymptomatic. Only RNLV1 RNA1 was found to replicate in N. benthamiana cells. We cloned the full-length RNLV1 RNA2 cDNA into the pCB301 vector and co-inoculated it with RNLV1 RNA1opt vector to N. benthamiana. Again, none of the inoculated plants showed virus symptoms. Western blot analysis showed that RNLV1 CP did accumulate in the inoculated N. benthamiana leaves, but not in their systematic leaves (data not shown). Therefore, whether RNLV1 and RNLV2 can infect whole plants remain unknown. It is possible that these two novel noda-like viruses are transmitted to rice plants in fields by unidentified insect(s). This insect vector(s) may also serve as the host(s) for virus replication. More field surveys and sample analyses are needed to address these unanswered questions.FHV has been shown to replicate in yeast, insect, plant, and mammalian cells ,30,61,67Due to their relatively small genomes, alphanodaviruses, especially FHV and NoV, have been modified and used as models in many studies ,30,31,32In summary, we have identified two novel noda-like viruses  in rice plants showing dwarf symptoms. Both viruses are clearly different from the reported nodaviruses, especially the CPs. These findings enrich our knowledge of nodaviruses and provide us with new virus-based vectors for future studies on the interaction between insect and plant. These findings can also help to develop more effective methods for rice virus disease management.


The global spread of human monkeypox disease, a zoonotic infection related to smallpox and endemic to West and Central Africa, presents serious challenges for health systems. As of July 2022, 14 533 cases have been reported world‐wide, leading to designation as a Public Health Emergency of International Concern. Monkeypox disease is spread from animals to humans through infected lesions or fluids; human–human transmission occurs through fomites, droplets or direct contact. Illness is usually self‐limiting, but severe disease can occur in specific groups ‐ particularly children, and people who are immunocompromised or pregnant. Clinical presentation may include fever, lymphadenopathy and skin rash, but the rash may occur without other symptoms. Complications can include secondary bacterial infection of skin lesions, vision loss from corneal involvement, pneumonia, sepsis and encephalitis. Diagnosis of monkeypox requires consideration of epidemiological, clinical and laboratory findings, with sensitive history‐taking, to elicit close contacts, critical. Supportive management is usually sufficient, but treatment options (where required) include antivirals and vaccinia immune globulin. A paucity of safety data for relevant antivirals may limit their use. There are two types of monkeypox vaccines: a replication‐competent vaccinia vaccine, the use of which is logistically and clinically complex, and a replication‐deficient modified vaccinia Ankara virus vaccine. Preparedness of health systems for addressing the current outbreak is constrained by historic underfunding for research, and compounded by stigma and discrimination against cases and affected communities. Key challenges in halting transmission include improving vaccine equity and countering discrimination against men who have sex with men to aid diagnosis and treatment. Monkeypox is a viral zoonotic disease that has been endemic in Central and West Africa for many decades. A global outbreak began in May 2022.It is caused by monkeypox virus, a member of the Orthopoxvirus genus, which also includes the smallpox (variola) and vaccinia viruses.Monkeypox is transmitted through close contact with an infected person or animal, or with material contaminated with the virus.Monkeypox disease is usually self‐limiting, but severe cases can occur, especially in pregnancy, children and immunocompromised patients.Smallpox vaccines can be used for pre‐exposure prophylaxis and post‐exposure prophylaxis against monkeypox disease, but are in limited supply and may be associated with serious adverse effects. An antiviral agent developed for the treatment of smallpox has also been licensed for the treatment of monkeypox.Monkeypox is a zoonotic disease capable of human‐to‐human transmission. It is caused by the monkeypox virus, an enveloped double‐stranded DNA virus. The virus belongs to the Orthopox genus of the Poxviridae family, shared by the smallpox (variola) virus and the vaccinia virus, which is used in smallpox vaccines.Whilst most monkeypox cases recover without severe consequences, the disease can cause significant morbidity and mortality in pregnancy, infancy and immunocompromised patients. Furthermore, monkeypox is considered a high‐consequence infectious disease, as it may be difficult to diagnose, is capable of person‐to‐person transmission and has limited effective prophylaxis and treatment options.The name ‘monkeypox’ stems from the virus's isolation from imported macaque monkeys in a Danish laboratory in 1958.Two distinct clades of the monkeypox virus have evolved, which vary in their clinical features and severity.When the smallpox eradication programme – which used first‐generation smallpox vaccines offering cross‐protection against monkeypox – ended successfully in 1980, the World Health Organization (WHO) established an active surveillance programme for monkeypox. This was discontinued in 1986, after predictions that future outbreaks would be self‐limiting.In enzootic countries, the exact reservoir species of monkeypox are unknown, but rodents are most likely.The clinical syndrome of monkeypox disease can be divided into two phases Table .17 AftMonkeypox is usually a self‐limiting disease with symptoms lasting from 2–4 weeks, but complications can occur in children, pregnant women and immunocompromised people.Although children are often significantly affected by monkeypox,To date, children aged under 10 years represent a large proportion of monkeypox‐related deaths, ranging from 100% of documented fatalities in the period 1970–1999 to 37.5% over 2000–2019.Congenital monkeypox infection has been described in one case series from the Democratic Republic of the Congo, with stillbirth a tragically common outcome.Recent European and UK guidance issued for the diagnosis and management of suspected monkeypox in pregnant women include the potential utility of available antivirals, vaccinia immune globulin (VIG) and safety considerations of post‐exposure vaccination.The diagnosis of monkeypox should consider the epidemiological, clinical and (if available) laboratory findings.The WHO recommends that health‐care workers looking after patients with suspected or confirmed monkeypox should observe contact and droplet precautions and wear appropriate personal protective equipment. If aerosol‐generating procedures are performed or varicella infection is suspected, the patient should also be cared for with airborne precautions.in vitro and challenge study data in animals.Smallpox vaccines using vaccinia virus are stockpiled due to the potential threat of smallpox bioterrorism, and these can also be used against monkeypox, for either pre‐exposure prophylaxis (PrEP) or post‐exposure prophylaxis (PEP). Evidence for the effectiveness of vaccinia vaccines specifically against monkeypox has been largely indirect, drawing on A live attenuated replication‐competent vaccinia vaccine (proprietary name ACAM2000), which is logistically challenging to administer, requiring percutaneous scarification using a bifurcated needle by trained vaccinators (for whom pre‐exposure vaccination to recommended to protect against inadvertent exposure to live vaccine virus);A replication‐deficient modified vaccinia Ankara virus vaccine  which is currently registered in North American and European countries for the prevention of smallpox and monkeypox.There are two types of smallpox vaccines:ACAM2000 is currently available in Australia for PrEP and PEP against monkeypox.By contrast, as the MVA‐BN vaccine is replication‐deficient (the live virus has been attenuated such that it is unable to replicate within cells), it is theoretically considered to be a safe vaccine for children, pregnant women and immunocompromised patients. Administration is by subcutaneous injection, which precludes autoinoculation with vaccine virus at other body sites. No serious safety concerns were identified during clinical trials in people receiving at least one dose of MVA‐BN, except one case of possible vaccine‐related pericarditis.Most cases of monkeypox are self‐limiting, with only supportive management needed. Hospital admissions in the current outbreak have occurred primarily to facilitate required isolation periods, manage severe anorectal or oropharyngeal pain, treat soft tissue superinfection, or administer therapies for severe disease.Treatments for monkeypox infections include antiviral agents and VIG, all of which are considered investigational for this indication.in vitro activity against monkeypox,Cidofovir is an antiviral agent registered for the treatment of cytomegalovirus in immunocompromised patients that has also been used against adenovirus and human herpesvirus.Finally, VIG – an intramuscular preparation of hyperimmune globulin prepared from the pooled blood of individuals vaccinated with smallpox vaccine – may be considered in immunosuppressed patients exposed to monkeypox, in whom ACAM2000 vaccine is contraindicated. VIG may also be used for aberrant infections induced by vaccinia virus due to autoinoculation, eczema vaccinatum, progressive vaccinia, or severe generalised vaccinia.Although monkeypox can infect all humans, concerns about stigma and discrimination associated with the current transmission of monkeypox in men who have sex with men are at the forefront of control measures and public communication. In many countries, under‐diagnosis of monkeypox is likely in the context of non‐heterosexual orientations being criminalised.Other forms of discrimination have included global inequities in access to vaccines, antiviral agents and laboratory testing capacity.Monkeypox is a high‐consequence infectious disease that, despite decades of endemicity in sub‐Saharan Africa, has only received widespread attention in 2022 with the emergence of a global outbreak affecting over 70 countries. Due to limited research into transmission, clinical severity and treatment options, the international community is poorly placed to prevent and treat this zoonotic disease despite ongoing human‐to‐human transmission and likely increased animal‐to‐human transmission, in the face of a changing global epidemiological climate. Alongside these challenges, the stigma and discrimination currently borne by patients infected with monkeypox impacts case ascertainment, and places vulnerable communities at increased risk of poor disease outcomes and continued transmission.


In the published article, there was an error in the article title. Instead of “Ocular effects caused by viral infections and corresponding vaccines: An overview of varicella zoster virus, measles virus, influenza viruses, hepatitis B viruses, and SARS-CoV-2,” it should be “Ocular effects caused by viral infections and corresponding vaccines: An overview of varicella zoster virus, measles virus, influenza viruses, hepatitis B virus, and SARS-CoV-2.”In the published article, there was an error. [Keywords: hepatitis B viruses].[Keywords]. This sentence previously stated:A correction has been made to “virus infection, vaccine, ocular effects, varicella zoster virus, measles virus, influenza viruses, hepatitis B viruses, SARS-CoV-2.”The corrected sentence appears below:“virus infection, vaccine, ocular effects, varicella zoster virus, measles virus, influenza viruses, hepatitis B virus, SARS-CoV-2.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.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.


Ascosphaera apis and some Aspergillus species are the main pathogenic fungi of honey bee, and A. apis is the pathogen of chalkbrood disease. However, the infection mechanism of them is incompletely known and it is still unclear whether other factors impact their pathogenesis. In this study, Aspergillus tubingensis were obtained from the chalkbrood bee samples for the first time. Our results showed that A. tubingensis could promote the accumulation of the spores of A. apis. Pathogenicity test found that inoculation of the spores of the two fungi alone or their combination could induce disease characterization of chalkbrood and stonebrood but the extent was less than those in field. To further identify other pathogens impacted the pathogenesis, we found several honey bee viruses presented in the pathogenic fungi A. apis and A. tubingensis, which were different from previous reported. Our results indicated that acute bee paralysis virus (ABPV) and chronic bee paralysis virus (CBPV) could replicate in these two fungi and increased in titer with the going of cultivation time. In addition, CBPV could not only transmit vertically to the next generation by spores, but also spread horizontally to different fungi through hyphal anastomosis. These results suggested that the honey bee chalkbrood contained the other pathogenic fungi besides A. apis, the interactions between different pathogens of chalkbrood microbial communities may influence the prevalence of chalkbrood. Moreover, the discovery of honey bee viruses and their transmission mode in these two fungi enhanced the potential of exploring fungi virus as valuable factors that cause fungal disease outbreak. Exoneura spp., Halictidae spp., and so on , acute bee paralysis virus (ABPV), Israeli Acute Paralysis Virus (IAPV), Kakugo virus (KV), and Deformed wing virus (DWV) were identified in A. tubingensis, while CBPV and ABPV in A. apis. It was also found that ABPV and CBPV not only replicated in A. tubingensis and A. apis, but also their titer was increased with the going of cultivation time. These studies provide a theoretical basis for the discovery of cryptic pathogenic fungi, and fungi as virus transmission vector and pathogen control in honey bees.Here, we speculated whether there were other “cryptic” fungi in chalkbrood, such as Apis mellifera L. were collected from the apiary severely infected with chalkbrood disease in Xinjiang province , China during April 2020. The chalkbrood mummies were collected from the bottom board or beehive entrance, while the surface of the mummies was covered with white filamentous and black spores. Then samples with typical symptoms from 10 colonies were selected, and all samples were stored in a refrigerator at −80°C until used for testing. Aspergillus niger ATCC16404 was purchased from China General Microbiological Culture Collection Center (CGMCC).Chalkbrood samples of Fungi were isolated and purified as previously described with modifications .Method 1 : Mummies were soaked in 10% NaClO for 10 min and washed twice in sterile water for 2 min. Then, the surface-sterilized chalkbrood mummies were cut into small pieces, which were further transferred to PDA  medium with 100 μg/ml of ampicillin and incubated for 3–6 days in the dark at 30°C. The fungi were purified using hyphal-tipping technique under microscope for two rounds and cultured on PDA medium without antibiotic.−2–10−4 and then spread on the PDA medium containing 100 μg/ml of ampicillin, which were incubated at 30°C for 2–3 days in darkness. Under the microscope, a single-spore colony was marked and the corresponding fungal plug was taken to new antibiotic-free PDA medium for further cultivation at 30°C for 4–6 days.Method 2 (single-spore separation): Surface sterilization of chalkbrood mummies was conducted as above described. Then honey bee mummies were chopped up with sterile scalpel and put into new tube containing sterile water. After vortex, spore suspension was gradiently diluted to 10The hyphae block of purified fungi with a diameter of 0.5 cm was cut at the edge of the colony and placed to the center of PDA medium covered by cellophane membrane. After cultivating at 30°C for 2–3 days, the marginal hyphae were cut and observed by microscope. Furthermore, the conidiophore and mature ascoma were gently picked with sterile tweezers and placed on a glass slide with two drops of sterile water, which were directly observed using microscope .Aspergillus; A. niger amplifications were used as positive controls. The PCR amplification was carried out in a thermocycler  under the following conditions: 2 min at 94°C, followed by 30 cycles at 94°C for 30 s, 55°C or 58°C for 30 s, and 72°C for 30 s, and 72°C, 5 min for extra extension. PCR products were electrophoresed in 1% agarose gel containing 1 mg/ml GoldView  and observed under gel imaging analysis system instrument . The target gene fragments were purified by gel extraction  and verified by DNA sequencing . The resulting sequences were analyzed by BLAST at https://blast.ncbi.nlm.nih.gov/Blast.cgi.Fungi with different morphology were further identified by amplification of conserved genes containing internal transcribed spacer (ITS) and β-tubulin  with 1 day old were collected from three random colonies located in the institute of apicultural research of Beijing, China during September and they were fed in sterile 48-well plate as proposed by Spore cysts were produced between two opposing mating types of escribed . Then thescribed . Briefly2O. PCR was performed with cycling conditions as follows: 94°C for 2 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and 72°C, 5 min for extra extension. Detection, sequencing, and analysis of the expected DNA products were performed as the above description. The sequence data from this study were submitted to and obtained accession numbers from GenBank.In order to detect several normal honey bee viruses in fungi, RNA extraction and reverse transcription PCR were conducted. For total RNA extraction, mycelial plugs were inoculated on PDA plates covered by sterilized cellophane membranes at 30°C for 6 days in the darkness. Around 100–200 mg mixture of mycelia and spores were collected and ground into a fine powder in liquid nitrogen. RNA extraction was conducted using ZN Fungal/Bacterial RNA Prep  according to the manufacturer’s instructions. The resulting RNA was stored at −80°C before needed. Synthesis of cDNA was performed with 1 μg of total RNA using HiFiscript cDNA Synthesis Kit  in the presence of primer mixture (consist of Oligo dT and random primer) according to the manufacturer’s instructions. Then RT-PCR reaction mixture contained 1 μl of forward and reverse primers , respectA. tubingensis and A. apis using ZN Fungal/Bacterial RNA Prep , and cDNA was synthesized using tagged specific primer , and the purified fragments were cloned into plasmid pMD-18T using pMDTM-18T Vector Cloning Kit . Then the constructed plasmids were transformed into competent cells Escherichia coli DH5α . The recombinant plasmids were extracted using the PurePlasmid MiniPrep Kit  and verified by sequencing . All the above reactions were performed according to the manufacturer’s recommended instructions. The resulting plasmids for RT-qPCR were diluted to 10−2–10−8 and reacted with specific forward or reserve primers and tag primer  and CBPV (OL321879) of A. tubingensis in this study. Primer sequence of tag was non-homologous to the honey bee virus as previously described  were used as templates. The other PCR reagents included non-tagged primer and tag primer  were similar to that of DNA amplification for standard curve. The reactions were performed on LineGene 9600 Plus real-time PCR instrument, and the results were analyzed using 9600 Plus Software and standard curves.In order to test whether virus can be transmitted to offspring or other related virus-free fungi, we followed the protocol of To study vertical transmission of viruses, fresh fungal plug was cultivated on PDA plate at 30°C for 5–7 days. When colonies produced conidia or ascospore, the solution of conidia or ascospore was prepared according to the above method. Then 100 μl of diluted spore suspension was spread on the PDA plate, which was incubated at 30°C for 2–4 days in darkness. The mycelia at the edge of a colony were collected for virus detection.A. niger through hyphal anastomosis. In brief, two different strains were placed on the two sides of PDA plate, respectively, with 2 cm distance. Ascosphaera tubingensis and A. apis served as the donors, whereas virus-free strain A. niger served as recipient. After 17 days under dark condition at 30°C, three mycelium plugs from A. niger were taken and placed on the new PDA plates. The mycelia near the edge middle of A. niger colony were labeled JL1, mycelia below the edge of A. niger colony were labeled JL2, and the mycelia far from A. tubingensis colony were labeled JL3. Detection of the viruses in mycelia using the methods above described.To study horizontal transmission of viruses, pairing culture technique was used. Viruses were transferred to virus-free strain of A. apis. Method of single-spore isolation was also adopted. After incubation for 2 days, a smaller special colony was observed , CBPV (accession number: OL321879), IAPV (accession number: OL321884), KV (accession number: OL321882), and DWV-A (accession number: OL321883) were detected in A. tubingensis. Besides, ABPV (accession number: MZ683936) and CBPV (accession number: OL321881) were detected in both A. apis HQS-1 and HQS-2. There was no difference in virus species between A. apis HQS-1 and HQS-2.The results of virus amplification in A. apis and A. tubingensis with very obvious bands, the replication and accumulation of which were tested in A. apis and A. tubingensis using strand-specific RT-qPCR. Hence, the presence of negative-strand RNA and positive-strand RNA for each virus would be determined when fresh fungal plugs were cultivated on PDA for 3, 6, and 9 days. The specificity of positive and negative chain amplification primers of ABPV and CBPV and the standard curves had been verified  could be detected in all spores, which may be beneficial to the long-term stable existence of the virus, while ABPV (12/19), IAPV (7/19), KV (5/19), and DWV-A (4/19) distributed in spores to varying degrees  could be also detected in all spores, while ABPV (10/15) distributed in partial spores  could infect an insect and use it as a transmission vector. Furthermore, it was also discovered that Cucumber mosaic virus (CMV) was transmitted between plants and fungi, and in the process of plant virus transmission, fungi were acted as vectors , and the above viruses belong to insect or fish viruses, plant viruses and fungal viruses, respectively  and Key Technology Research and Development Program of Guangxi province (AB16380094).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.


Herpes simplex virus (HSV) causes chronic infection in the human host, characterized by self-limited episodes of mucosal shedding and lesional disease, with latent infection of neuronal ganglia. The epidemiology of genital herpes has undergone a significant transformation over the past two decades, with the emergence of HSV-1 as a leading cause of first-episode genital herpes in many countries. Though dsDNA viruses are not expected to mutate quickly, it is not yet known to what degree the HSV-1 viral population in a natural host adapts over time, or how often viral population variants are transmitted between hosts. This study provides a comparative genomics analysis for 33 temporally-sampled oral and genital HSV-1 genomes derived from five adult sexual transmission pairs. We found that transmission pairs harbored consensus-level viral genomes with near-complete conservation of nucleotide identity. Examination of within-host minor variants in the viral population revealed both shared and unique patterns of genetic diversity between partners, and between anatomical niches. Additionally, genetic drift was detected from spatiotemporally separated samples in as little as three days. These data expand our prior understanding of the complex interaction between HSV-1 genomics and population dynamics after transmission to new infected persons. Herpes simplex virus 1 (HSV-1) causes chronic oral and genital infections for which there are currently no vaccines, and epidemiology trends indicate that an increasing number of primary infections occur in the genital anatomical niche. We provide a clinical genomics analysis of adult sexual transmission of HSV-1, in the context of new genital infections. We find that viral genomes are mostly conserved between transmission partners, while still exchanging within-host diversity between partners. We showed that this diversity can persist between distinct periods of viral reactivation, and involve multiple viral genotypes. This study significantly improves our understanding of the sources of HSV-1 genetic diversity, which will be critical in designing effective therapeutics against the wide range viral genotypes and disease severity. Human alphaherpesvirus 1; family Herpesviridae, subfamily Alphaherpesviridae) is a highly prevalent human pathogen that infects over 60% of the global population and causes a wide range of symptom severity  or at insertion and deletion sites [i.e. in/dels]). This included one or more consensus-level SNVs (outside of those at repetitive elements or in/dels) between the viral genomes in three out of five transmission pairs . Since these differences are not easily visualized in the larger network graph , we purs7 (reference strain) to T8 within the gene encoding glycoprotein H , which removes a stop codon , with the exception of indels at repetitive sites in the genome. Their shared nucleotide identity included a homopolymer frameshift mutation of Top codon . This T7no acids . Only onSeveral previous studies applying deep-sequencing of both cultured and uncultured HSV-1 samples have detected varying levels of within-host diversity, or minor variants (MVs), within a single sample from an infected host. These MVs indicate within-host variation, or the potential presence of multiple virus genotypes –9,30. WeA greater level of within-host diversity and MVs was observed in viral genomes isolated from Pair 1 (v40 and v41), Pair 4 (v46 and v47), and Pair 5 (v48) . Pairs 1We next explored the presence of coincident MVs in transmission partners, indicating potential transfer of viral populations during initial infection. As noted above, both partners in Pair 2 (v42 and v43) enrolled with primary genital infection, and thus the directionality of their transmission cannot be known with certainty. The transmission event that resulted in Pair 2 occurred within one month of study enrollment for both participants , which aNext, we compared viral genetic diversity present within overall genital-area samples versus site-specific genital lesion samples in the same shedding episode, to better understand the localization and/or spread of viral variants within an anatomical niche. A second minor variant elsewhere in the UL27 gene encoding gB was detected within v43 samples. In this case the nucleotide difference encoded a missense mutation in gB . This minor variant reached a frequency sufficient to create a consensus-level difference (a SNV) between same-day swabs of the whole genital area, vs. site-specific genital lesion samples . In the Samples from Pair 2 also illustrate other potential sites of shared within-host diversity. For instance, the v43 samples from day 349 also harbor a minor variant in the UL2 gene, which encodes uracil-DNA glycosylase. In the genital area sample v43_d349_gen, there is a minor variant detected at a ratio of 95% T to 5% G (a silent variant at AA77 in UL2). In the site-specific genital lesion from the same day (v43_d349_gen_les), the ratio of nucleotides is 40% T to 60% G at this location in the UL2 gene, echoing the frequencies of the UL27 (gB) variants in the same samples . The parIn this study, we were able to compare the presence or absence of within-host viral genetic diversity between partners and across anatomical niches. In transmission Pair 1, the source partner, participant 40, reported first symptoms of oral HSV-1 infection 14 years prior to transmission of HSV-1 to the recipient partner, participant 41. We sequenced three samples from participant 40 , and two samples from participant 41 . In the source v40 genomes, within-host diversity was only detectable in the first of two oral samples  with 27 MVs identified and all of those being below 4% frequency . Of the To explore potential co-segregation and transmission of MVs in Pair 1, we compared the position and identity of each MV to check for shared variants between both participants. In addition to looking at within-host MVs detected in both participants, we also considered whether a variant detected in one participant might be present in the other partner, but be below our minimum threshold for detection. This is especially relevant due to the lower coverage of the v41 oral sample (28X). This analysis indicated that all of the variants detected in source v40 genome (n = 27) were also detectable in recipient v41 genome, either above or below the adjusted threshold for MV detection . Similar10 copies per mL). Therefore, we pooled four genital swab samples spanning two shedding episodes within the same month, creating v47_d61-79_gen  as the new host’s immune response develops. Our data describe five transmission pairs, in which we compared viral genome sequences sampled from viral shedding in both the oral and genital niches over a one-year period. Each set of transmission-pair viral genomes occupied a unique branch in a network graph containing 65 other HSV-1 genomes. We found ≥ 98% nucleotide identity between viral genomes at the consensus-level within each transmission pair. We also noted single nucleotide differences (or SNVs), occurring at the consensus level in three out of five pairs . Several of these differences arose over distinct shedding episodes, while others fluctuated within a single episode, as detected in participant 43’s genital samples (Pair 2). Across all participants and specimen types, we found within-host viral genetic diversity to be limited and often transient. The quantity of within-host diversity, or minor variants (MVs), was similar between transmission partners, although in several cases MVs were differentially detected between site-specific lesion samples vs. those encompassing the entire genital area. These data reveal that consensus-level nucleotide changes can occur after transmission of genital HSV-1, and that minor variant frequencies can fluctuate over time and between shedding episodes. The ability for HSV-1 to undergo genetic drift and/or selection during transmission is likely driven by the extent of within-host diversity present during a particular shedding episode, as well as viral genetic fitness with respect to each host.When HSV-1 infections have been sampled from unrelated individuals, we and others have found these to be genetically distinct ,10,23,30in utero have suggested a population bottleneck exists during transmission, followed by population expansion in neonatal niches . Al. Al31]. 10 copies per mL) have generally yielded insufficient viral DNA using current sequencing methods, thus we prioritized samples with higher copy numbers. We also limited sample selection to those that would optimize sequencing cost and effort while spanning multiple shedding episodes . In the case of participant 47, no samples were above 10,000 genome copies, so we pooled four samples to create a baseline consensus viral genome for this individual , as in prior studies of genital HSV shedding ,53. For dividual . For sevdividual . Each paSwabs selected based on the criteria above were processed for target enrichment and sequencing according to our published protocol . Briefly−2 were retained. These PS reads were then processed through the first step of the Viral Genome Assembly (VirGA) pipeline for quality control  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Ekaterina E. HeldweinGuest EditorPLOS PathogensErik FlemingtonSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Both herpes simplex virus 1 and 2 (HSV-1 and HSV-2) can be responsible for genital herpes lesions. HSV-1 classically oral infections and HSv-2 genital infections, however HSV-1 epidemiology is evolving in favour of an increasing number of primary infections occur in the (ana-)genital anatomical niche. Viruses could adapt to their host over time thanks to genetic adaptation. As few data are known for HSV, especially for HSV-1 in the context of genital herpes, all studies concerning this subject are quite interesting. Then, the authors, Rathbun at al., try to study viral genetic diversity in the context of HSV-1 genital herpes. Indeed, they used deep-sequencing analysis to characterize the consensus HSV-1 sequences and to detect minor variants (MVs) within paired sexual partner samples.Reviewer #2: Rathbun et al describe an important and elegant study of genetic variation in herpesvirus genomes among recently infected sexual partners. Both the study and the manuscript are highly accomplished and the authors are to be congratulated.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: I have read through the manuscript and I have major comments.- To my opinion, the most bothering fault of this study is the analysis of sequencing data. Indeed how is it possible to take into account sequencing data whereas average coverage is very low . The use of threshold of 20% supposedly more stringent is not appropriated. To my opinion, to be relevant, only samples with coverage depth <100x should not be considered. Most of the time, low average coverage is associated few reads available and high MV detection: e.g. Sample v47_d61-79_gen with 31x of average coverage associated with 225 MVs or Sample v41_d354_oral with 28x of average coverage associated with 144 MVs.Can we trust that?Moreover, the authors do not discuss quality criteria for sequence trimming in the MM section. May the authors cite publications where these criteria have been used for NGS analysis for HSV genomic diversity estimation?- According to the authors, detection of MVs within the viral population revealed both shared and unique patterns of genetic diversity between partners, and between anatomical niches. But how to make the clear distinction anal and genital niche with self-sampling samples.- Additionally, the authors discuss that genetic drift was detected from spatiotemporally separated samples in as little as three days. This period of time of 3 days appear to be unrealistic. Indeed, even under antiviral selection pressure for instance the evolution pf HSV-1 do not reach such a level of MV within viral genes implicated in antiviral resistance.- It appears there is some discordance between text/tables/figure data makes difficult the interpretation of the study. Either I do not understand or this formulation is not clear enough for well-understanding! I think the authors simplify the tables and the figures to get straight to the point.Reviewer #2: (No Response)**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: NONEReviewer #2: There are a number of places where it appears the text is referring to the wrong figure number or sub-number eg 6b rather than 6 C on page 13 line 292. It would be useful for the authors to systematically review all such references for accuracy.The discussion should include comments on the limitations of the methodology in terms of variation in the depth of sequencing coverage between samples and the implications of this on the ability to detect MV.**********what does this mean?). 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Read more information on sharing protocols at  9 Mar 2022AttachmentResponse_to_Review_PLoS_Path_transmission_paper_v7.pdfSubmitted filename: Click here for additional data file. 11 Mar 2022Dear Dr. Szpara,We are pleased to inform you that your manuscript 'Comparison of herpes simplex virus 1 genomic diversity between adult sexual transmission partners with genital infection' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. 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Hepatitis A virus (HAV) is one of the well-known viruses that cause hepatitis all around the globe. Although this illness has decreased in developed countries due to extensive immunization, numerous developing and under-developed countries are struggling with this virus. HAV infection can be spread by oral-fecal contact, and there are frequent epidemics through nutrition. Improvements in socioeconomic and sanitary circumstances have caused a shift in the disease's prevalence worldwide. Younger children are usually asymptomatic, but as they become older, the infection symptoms begin to appear. Symptoms range from slight inflammation and jaundice to acute liver failure in older individuals. While an acute infection may be self-limiting, unrecognized persistent infections, and the misapplication of therapeutic methods based on clinical guidelines are linked to a higher incidence of cirrhosis, hepatocellular carcinoma, and mortality. Fortunately, most patients recover within two months of infection, though 10–15% of patients will relapse within the first six months. A virus seldom leads to persistent infection or liver damage. The mainstay of therapy is based on supportive care. All children from 12–23 months, as well as some susceptible populations, should receive routine vaccinations, according to the Centers for Disease Control and Prevention and the American Academy of Pediatrics. Laboratory diagnosis of HAV is based on antigen detection, checking liver enzyme levels, and antibody screening. Furthermore, polymerase chain reaction (PCR) technology has identified HAV in suspected nutrition sources; therefore, this technique is used for preventative measures and food-related laws. Acute viral hepatitis is a systemic illness that mainly affects the liver. Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis D virus (HDV), and hepatitis E virus (HEV) are the viruses that cause almost all instances of acute viral hepatitis . HepatitIn this perusal, we look at HAV, initially discovered in the feces of tentatively infected human prisoner volunteers using electron microscopy . HAV infSporadic or epidemic outbreaks of HAV even now arise in several parts of the world, particularly in developing nations. In regions where people are mostly vaccinated, the prevalence of HAV has dropped considerably. In more industrialized nations, there is a growing movement toward vaccinating children, as this minimizes the chance of infection in the rest of the population. A prominent event occurs in countries with median endemicity when the disease is rare due to the amended water treatment and hygiene. However, grown-ups with no earlier contact become infected through sporadic outbreaks with clinically apparent symptoms , 15. SymThe purpose of this study is to examine the existing evidence for hepatitis A screening, diagnosis, and treatment. The structure of the HAV virus, the virus genome, how it is diagnosed, how it spreads, the importance of vaccination, and the factors derived from the HAV virus are all discussed.Every year, millions of HAV cases are recorded, with a more significant occurrence in underdeveloped countries . The WorOver 90% of children experience the infection before the age of ten. HAV infection is asymptomatic in around 70% of kids under six, with the majority showing no clinical indications of liver disease, while symptoms appear in 70% of adolescents and adults. In adults, HAV can cause severe morbidity, with jaundice occurring in 40–70% of cases .HAV is a tiny virus with a diameter of 27–32 nm . HAV is Infectious viruses come in two types: naked, nonenveloped HAV virions, which are released into the stool, and quasienveloped virions (eHAV), created by seizing control of the host. Endosomal sorting complexes required for transport (ESCRT) by HAV and nonlytic viruses are produced from infected cells, circulating in the bloodstream as a membrane-cloaked complex or in the supernatant of infected cell cultures during acute infection, and then complexes can be produced , 31. TheHepatovirus genus within the family Picornaviridae, including several other medical and veterinary viruses. Although HAV shares many characteristics with other Picornaviridae family members, its partial nucleotide sequence and numerous unique features set it apart from other picornaviruses, requiring it to be assorted into its genus. HAV strains obtained from nonhuman primates and the ones recently found in bats, hedgehogs, shrews, and rodents appear to share antigenic elements with human HAV and are among the viruses designated as hepatoviruses [HAV is classified as a member of the oviruses –28. HAV oviruses , 38. Genoviruses  , known as HAV cellular receptor 1 (HAVCR1), was once thought to be the HAV receptor . HoweverHAV replicates in the liver  and peneHAV is a long-lasting virus with many properties, such as genomic organization and a long replication cycle, distinguishing it from the rest of the mammalian picornaviruses. HAV proteins are produced via cap-independent translation of a singular, long open reading frame under the control of an ineffective upstream IRES. Genome replication is slow and noncytopathic, as in picornaviruses, and transcription is presumably carried out by a uridylated protein primer . BecauseIn infected cells with various viruses, including HBV and HCV, the cooxygenase-1 (HO-1) enzyme exhibits antiviral properties . StudiesTo liberate its membrane-cloaked eHAV form, HAV can seize an apoptosis-linked gene 2-interacting protein X (ALIX)-related exosome-like pathway . HAV repHAV infects vulnerable individuals, multiplying in the liver, and the newly reproduced virus is released into the intestines, where it is excreted in feces . Acute hHAV can induce severe clinical hepatitis and death in rare cases, but persistent infection or chronic hepatitis is not familiar, even in highly immunocompromised patients . While sIn the laboratory, clinical specimens such as blood, feces, bile, liver biopsy, and serum are utilized to detect HAV. The following items are some of the most common procedures for hepatitis A laboratory diagnosis:(1) Antibody detection: HAV-specific IgM antibodies are typically found in the earliest stage of disease and can last for six months after infection. IgG antibodies can last for many years. Enzyme-linked immunosorbent assay (ELISA) is used to distinguish antibodies. Rapid tests (based on immunochromatographic technologies) can also be used to detect antibodies. (2) Liver enzyme levels: measurement of liver enzyme levels such as ALT, alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGTP), and serum bilirubin. (3) Antigen detection: PCR and nucleic acid hybridization assays are used to identify antigens. Also, conventional testing for HAV relies on anti-HAV IgM seropositivity. However, studies estimate that 10–30% of patients may not be diagnosed by serology. Molecular assays that can directly detect viral nucleic acids have the potential to improve diagnosis , which is key to preventing the spread of infections –87 Figu. AntibodHAV identification in food has been accomplished by utilizing molecular tests with fit-for-purpose primers to provide 100% specificity. Although conventional PCR is still valid since more oversized PCR products are generated to validate the target's specificity, quantitative RT-PCR (RT-qPCR) technologies have positively altered viral identification in medical and environmental samples. The high sensitivity of RT-qPCR, its low cost, and the increased specificity achieved by using fluorescent probes have made it the gold standard for HAV detection in food. RT-qPCR, in particular, allows for quantitative viral detection for threat risk assessment analysis and is applied to enhance preventative measures and food-related laws , 88. WhiWhen people are infected with HAV, it is medically impossible to distinguish the infection from other types of acute viral hepatitis, and the disease is generally mild and self-limiting. Elderly or immunocompromised people suffering from chronic liver disease or other underlying health problems are in danger of developing symptoms . EnvironCampylobacter, influenza virus, Ebstein–Barr virus, and cytomegalovirus (CMV) infections being the most prevalent [Guillain–Barré syndrome (GBS) is a peripheral demyelinating illness frequently followed by a gastrointestinal infection. The infectious cause is mainly bacterial or viral, with revalent . Even threvalent , 93. Altrevalent . False-prevalent . Also, crevalent . As a rerevalent .During the last few decades, blood safety has grown in importance as a means of preventing diseases associated with transfusions . TransfuInfection with HAV can cause the development of antibodies against the virus's structural and nonstructural polypeptides. In contrast, an inactivated or attenuated HAV vaccine produces antibodies primarily against structural proteins, with little or no antibody production against nonstructural proteins . HepatitPeople at risk of contracting the virus should be vaccinated. People with homosexual tendencies or travelers to countries with high or moderate HAV rates and those consuming illegal substances or suffering from chronic liver disease, as well as those in jail, emergency rooms, syringe exchange programs, and drug treatment facilities, are among the most vulnerable , 103. FoEscherichia coli expression system for recombinant synthesis of one of the vital capsid proteins, VP1, from HAV. VP1 oligomers produced in a bacterial expression system can be used to study the molecular mechanism of HAV capsid formation and may have biological applications in preventing HAV infections [The existing HAV vaccines depend on generating wild-type or attenuated virus in cell culture, which increases production costs . The crefections . Nanoparfections . Numeroufections . In recefections . Immune fections . Long-tefections . Anti-HAfections . Serologfections .Although common sexually transmitted disease (STD) prevention techniques  are insufficient to avoid HAV transmission, they are a vital aspect of education for the MSM and the population who are engaged with high-risk sexual behaviors. Functional vaccination is available and safe, so its usage is advised to avoid completely transmitted infections and outbreaks . AdditioOne approach to avoiding HAV infection is nutrition, which can inhibit HAV growth and replication. As previously stated, higher GRP78 expression inhibits HAV replication . JapanesDespite the accessibility and effectiveness of hepatitis A vaccinations, hepatitis A outbreaks continue to be reported globally. Creating antiviral medication is significantly challenging. Antiviral-based medication may be helpful in preventing and treating fulminant hepatitis, shortening the infective phase, and decreasing the severity of symptoms, hence lowering the likelihood of outbreaks and the transmission of vaccine-escape mutations , 115. DiAs addressed in this article, HAV is still a serious concern in developing countries. The epidemiology of HAV infection should be monitored constantly to reduce the risks of any outbreaks, which could be achieved by undertaking cost-effectiveness analyses of HAV immunization techniques. Also, immunization has to be considered an important step, and this system will enable the early detection of epidemiologic transitions and the implementation of preventative efforts before HAV infection becomes a public health issue. Owing to the importance of this virus, further relevant studies are crucial to revealing unknown aspects of the HAV entrance mechanism to amend the lack of appropriate therapeutic medication.