Negative Stain Electron Microscopy Research Articles

Revisiting sodium phosphotungstate and ammonium molybdate as nonradioactive negative-staining agents for single-particle analysis.

This study reports the successful replacement of uranyl-based stains by either sodium phosphotungstate or ammonium molybdate in negative-staining electron microscopy. Using apoferritin as a test specimen, it is demonstrated that in combination with a facile on-grid fixation step, both stains yield comparable images to uranyl formate. Subsequently, using β-galactosidase, it is shown that both stains can also successfully be employed for single-particle analysis, yielding virtually indistinguishable results from uranyl formate. As both replacement stains are nonradioactive, they are not subjected to the same handling restrictions as uranyl-based stains. Therefore they are not only cheaper to use, but also make decentralized sample-grid preparation, directly after purification, accessible to a broader range of scientists.

Diagnostic electron microscopy in human infectious diseases - Methods and applications.

Diagnostic electron microscopy (EM) is indispensable in all cases of infectious diseases which deserve or profit from the detection of the entire pathogen (i.e. the infectious unit). The focus of its application has shifted during the last decades from routine diagnostics to diagnostics of special cases, emergencies and the investigation of disease pathogenesis. While the focus of application has changed, the methods remain more or less the same. However, since the number of cases for diagnostic EM has declined as the number of laboratories that are able to perform such investigations, the preservation of the present knowledge is important. The aim of this article is to provide a review of the methods and strategies which are useful for diagnostic EM related to infectious diseases in our days. It also addresses weaknesses as well as useful variants or extensions of established methods. The main techniques, negative staining and thin section EM, are described in detail with links to suitable protocols and more recent improvements, such as thin section EM of small volume suspensions. Sample collection, transport and conservation/inactivation are discussed. Strategies of sample examination and requirements for a proper recognition of structures are outlined. Finally, some examples for the actual application of diagnostic EM related to infectious diseases are presented. The outlook section will discuss recent trends in microscopy, such as automated object recognition by machine learning, regarding their potential in supporting diagnostic EM.

Rational design of uncleaved prefusion-closed trimer vaccines for human respiratory syncytial virus and metapneumovirus.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause human respiratory diseases and are major targets for vaccine development. In this study, we design uncleaved prefusion-closed (UFC) trimers for the fusion protein (F) of both viruses by examining mutations critical to F metastability. For RSV, we assess four previous prefusion F designs, including the first and second generations of DS-Cav1, SC-TM, and 847A. We then identify key mutations that can maintain prefusion F in a native-like, closed trimeric form (up to 76%) without introducing any interprotomer disulfide bond. For hMPV, we develop a stable UFC trimer with a truncated F2-F1 linkage and an interprotomer disulfide bond. Dozens of UFC constructs are characterized by negative-stain electron microscopy (nsEM), x-ray crystallography (11 RSV-F structures and one hMPV-F structure), and antigenic profiling. Using an optimized RSV-F UFC trimer as bait, we identify three potent RSV neutralizing antibodies (NAbs) from a phage-displayed human antibody library, with a public NAb lineage targeting sites Ø and V and two cross-pneumovirus NAbs recognizing site III. In mouse immunization, rationally designed RSV-F and hMPV-F UFC trimers induce robust antibody responses with high neutralizing titers. Our study provides a foundation for future prefusion F-based RSV and hMPV vaccine development.

Lysosomal enzyme binding to the cation-independent mannose 6-phosphate receptor is regulated allosterically by insulin-like growth factor 2.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. CI-MPR is multifunctional and modulates embryonic growth and fetal size by downregulating circulating levels of the peptide hormone insulin-like growth factor 2 (IGF2). The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. How a particular ligand affects the receptor's conformation or its ability to bind other ligands remains poorly understood. To address these questions, we purified a soluble form of the receptor from newborn calf serum, carried out glycoproteomics to define the N-glycans at its 19 potential glycosylation sites, probed its ability to bind lysosomal enzymes in the presence and absence of IGF2 using surface plasmon resonance, and assessed its conformation in the presence and absence of IGF2 by negative-staining electron microscopy and hydroxyl radical protein footprinting studies. Together, our findings support the hypothesis that IGF2 acts as an allosteric inhibitor of lysosomal enzyme binding by inducing global conformational changes of CI-MPR.

A multispecific antibody against SARS-CoV-2 prevents immune escape in vitro and confers prophylactic protection in vivo.

Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trivalent trispecific antibody that potently neutralized diverse SARS-CoV-2 variants and prevented virus escape more effectively than single antibodies or mixtures of the parental antibodies. Despite being generated before the appearance of Omicron, this trispecific antibody neutralized all major Omicron variants through BA.4/BA.5 at nanomolar concentrations. Negative stain electron microscopy suggested that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated binding across more than one spike protein. Moreover, a tetravalent trispecific antibody containing the same variable regions as the trivalent trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2, and BA.5 challenge, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. These results demonstrated that multispecific antibodies have the potential to provide broad SARS-CoV-2 coverage, decrease the likelihood of escape, simplify treatment, and provide a strategy for antibody therapies that could help eliminate pandemic spread for this and other pathogens.

7222 Structural and Mechanistic Studies of the Bioactive Anti-Müllerian Hormone Procomplex

Abstract Disclosure: J.A. Howard: None. L. Hok: None. N.J. Sanford: None. R.L. Cate: None. K. Hart: Employee; Self; Regeneron Pharmaceuticals. E. Leach: None. D. Pepin: None. P.K. Donahoe: None. T.B. Thompson: Advisory Board Member; Self; Oviva Therapeutics, Keros Therapeutics. The Anti-Mullerian hormone (AMH), a divergent member of the TGF-beta signaling superfamily, plays crucial roles in reproductive development and maintenance. Owing to its key role as a negative feedback regulator in folliculogenesis, AMH has garnered significant interest for therapeutic applications in contraception and oncofertility. The bioactive form of AMH is a noncovalent complex, comprising the TGFβ-like signaling domain and a dimeric prodomain whose structure remains largely unexplored. The prodomain component is necessary for the dimerization and secretion of the signaling domain, shuttling the ligand to autocrine, paracrine, and endocrine targets. In contrast, the structure and biochemistry of this domain remain poorly characterized, and its role in enhancing AMH signaling remains unclear. This gap in understanding significantly limits the enhancement and wider application of AMH-based protein therapeutics. Molecular modeling unveiled an unexpected AMH prodomain structure: an N-terminal dimerizing domain (NTD) linked to a novel C-terminal binding domain (CTD). This atypical design was affirmed by both small-angle X-ray scattering and negative-stain EM. The CTD introduces a unique TGFβ superfamily fold consisting of a four-helix bundle and an unstructured binding “belt”. The more prodomain-like NTD does not interact with the signaling ligand, nor does it consistently interact with the CTD. Both domains exhibit high levels of interspecies conservation as well as disease-causing mutations, such as those found in PMDS, PCOS, and POI. Using single-particle Cryo-EM we have elucidated the molecular mechanisms of the interaction between the AMH prodomain and signaling domain bound by mAb 6E11. Our 3.2Å cryo-EM structure confirmed the predicted architecture and binding function of the CTD and demonstrated a new dimension of conformational plasticity within the signaling ligand. We observed movements up to 5Å at the fingertips and 10Å in the outer helices of the CTD. More unexpectedly, the conformation of the AMH signaling ligand in the procomplex form is far more open and extended than has been observed in any other structure of a TGF-beta superfamily ligand, including the AMH ligand bound to its receptor AMHR2. We see that transitioning from a prodomain-bound to a receptor-bound state involves significant conformational remodeling of the signaling domain. This reorganization supports a conformational shift mechanism for AMH signaling in which bivalent binding of AMHR2 at the cell surface induces a >15Å and 38° closing of the ligand fingertips, leading to prodomain displacement and subsequent recruitment of type I receptors. Our findings correct earlier structural misconceptions and build a comprehensive model for interpreting existing functional data. This structure provides a foundation for understanding AMH dysregulation and developing advanced AMH therapeutics. Presentation: 6/1/2024

DNA-Based Molecular Clamp for Probing Protein Interactions and Structure under Force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology, and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA-based device in which the transition between single- and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension, while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Modular Structure and Polymerization Status of GABAA Receptors Illustrated with EM Analysis and AlphaFold2 Prediction.

Type-A γ-aminobutyric acid (GABAA) receptors are channel proteins crucial to mediating neuronal balance in the central nervous system (CNS). The structure of GABAA receptors allows for multiple binding sites and is key to drug development. Yet the formation mechanism of the receptor's distinctive pentameric structure is still unknown. This study aims to investigate the role of three predominant subunits of the human GABAA receptor in the formation of protein pentamers. Through purifying and refolding the protein fragments of the GABAA receptor α1, β2, and γ2 subunits, the particle structures were visualised with negative staining electron microscopy (EM). To aid the analysis, AlphaFold2 was used to compare the structures. Results show that α1 and β2 subunit fragments successfully formed homo-oligomers, particularly homopentameric structures, while the predominant heteropentameric GABAA receptor was also replicated through the combination of the three subunits. However, homopentameric structures were not observed with the γ2 subunit proteins. A comparison of the AlphaFold2 predictions and the previously obtained cryo-EM structures presents new insights into the subunits' modular structure and polymerization status. By performing experimental and computational studies, a deeper understanding of the complex structure of GABAA receptors is provided. Hopefully, this study can pave the way to developing novel therapeutics for neuropsychiatric diseases.

Design and immunogenicity of an HIV-1 clade C pediatric envelope glycoprotein stabilized by multiple platforms.

Various design platforms are available to stabilize soluble HIV-1 envelope (Env) trimers, which can be used as antigenic baits and vaccine antigens. However, stabilizing HIV-1 clade C trimers can be challenging. Here, we stabilized an HIV-1 clade C trimer based on an Env isolated from a pediatric elite-neutralizer (AIIMS_329) using multiple platforms, including SOSIP.v8.2, ferritin nanoparticles (NP) and an I53-50 two-component NP, followed by characterization of their biophysical, antigenic, and immunogenic properties. The stabilized 329 Envs showed binding affinity to trimer-specific HIV-1 broadly neutralizing antibodies (bnAbs), with negligible binding to non-neutralizing antibodies (non-nAbs). Negative-stain electron microscopy (nsEM) confirmed the native-like conformation of the Envs. Multimerization of 329 SOSIP.v8.2 on ferritin and two-component I53-50 NPs improved the overall affinity to HIV-1 bnAbs and immunogenicity in rabbits. These stabilized HIV-1 clade C 329 Envs demonstrate the potential to be used as antigenic baits and as components of multivalent vaccine candidates in future.

Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme binding.

Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in P. aeruginosa clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.

Unraveling the structure and function of a novel SegC protein interacting with the SegAB chromosome segregation complex in Archaea.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

Swinepox virus: an unusual outbreak in free-range pig farms in Sicily (Italy)

BackgroundTwo outbreaks of swinepox were investigated in free-range domestic pig farms located in the northeastern side of Sicily, Italy. The disease is generally self-limiting with a low mortality rate, but morbidity can reach high rates in case of poor sanitary conditions, improper husbandry practices and ectoparasitic infestation. The presented cases are the first ever reported on the island and part of the few cases reported in domestic pigs.Case presentationCarcasses condemned at the slaughterhouse and deceased pigs from Farm A and Farm B respectively, were referred for post-mortem examination and further investigations, with a strong suspect of SwinePox virus (SWPV) infection. Twelve deceased pigs were examined in total, showing poor body condition and pustular lesions scattered all over the cutaneous surfaces. Moreover, pigs from Farm B showed ocular lesions classified from Grade I to IV (from mild conjunctivitis to severe keratoconjunctivitis with corneal oedema, opacity, and ulcers). Final diagnosis was pursued by the microscopic assessment of skin lesions in both farms, which revealed the typical SWPV-lesion appearance, such as severe and disseminated ulcerative dermatitis and suspected inclusion bodies multifocally observed in the epidermis. Moreover, negative staining Electron Microscopy (nsEM) was performed on skin lesions and ocular swabs from Farm B, revealing in two samples the presence of brick-shaped viral particles, 220 nm long and 160 nm wide, with irregularly arranged surface tubules, identified as SWPV. The gene encoding the 482-bp fragment of the virus late transcription factor–3 was detected by PCR and sequencing revealed 99.79% identity and 100% query-cover with a strain previously isolated in Germany. Field clinical assessment was then performed in Farm B, revealing high overcrowding, poor sanitary conditions and improper husbandry practices, which are relevant risk factors for SWPV transmission.ConclusionsThe present is the first case report of SWPV in free-range pigs raised in Sicily, an island of the Southern coast of Italy, and wants to raise awareness on a neglected disease, and cause of animal health and welfare issues.

Formation and survival of clay-mediated protocell membranes composed of single-chain amphiphiles in aqueous solutions

Protocell compartments composed of simple amphiphilic molecules could have survived on the clay mineral surfaces which can serve well as plausible sites for prebiotic chemistry on the early Earth. Herein we chose four common single-chain amphiphiles (SCAs), including the anionic surfactant sodium dodecyl sulfate (SDS) and sodium dodecylbenzenesulfonate (SDBS), the cationic surfactant dodecyltrimethylammonium bromide (DTAB) and the zwitterionic lauryl sulfobetaine (LSB)), as model constituents of early membranes to establish a tentative model for protocell compartment. Positively charged Mg2Al-/Mg2Fe-layered double hydroxides (LDHs) and FeS2 are viewed as models of clay minerals. We demonstrate by comprehensively experimental study that unconventional vesicles can be easily promoted in a SCA aqueous micellar solution in the presence of model particles. Morphology evolutions are traced by collecting samples from mixtures of LDHs and SCA aqueous solutions and then recording at different incubation times via negative-staining or cryo-transmission electron microscopy (NS-TEM or cryo-TEM) and dynamic light scattering (DLS). Additionally, the stability of SCA bilayer membranes under possible prebiotic environment are tentatively studied. This work not only offers a robust yet versatile approach for the fabrication of novel vesicles but also provides new insights into the origin of cellular life.

In vitro higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs.

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.

Structural Mapping of Polyclonal IgG Responses to HA After Influenza Virus Vaccination or Infection.

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing context for future vaccine trials and emphasizing the importance of carefully designing vaccines to boost protective responses towards conserved epitopes.

Elasticity and Thermal Stability are Key Determinants of Hearing Rescue by Mini-Protocadherin-15 Proteins.

Protocadherin-15 is a core protein component of inner-ear hair-cell tip links pulling on transduction channels essential for hearing and balance. Protocadherin-15 defects can result in non-syndromic deafness or Usher syndrome type 1F (USH1F) with hearing loss, balance deficits, and progressive blindness. Three rationally engineered shortened versions of protocadherin-15 (mini-PCDH15s) amenable for gene therapy have been used to rescue function in USH1F mouse models. Two can successfully or partially rescue hearing, while another one fails. Here we show that despite varying levels of hearing rescue, all three mini-PCDH15 versions can rescue hair-cell mechanotransduction. Negative-stain electron microscopy shows that all three versions form dimers like the wild-type protein, while crystal structures of some engineered fragments show that these can properly fold and bind calcium ions essential for function. In contrast, simulations predict distinct elasticities and nano differential scanning fluorimetry shows differences in melting temperature measurements. Our data suggest that elasticity and thermal stability are key determinants of sustained hearing rescue by mini-PCDH15s.

Midkine Attenuates Aβ Fibril Assembly and AmyloidPlaque Formation.

Proteomic profiling of Alzheimer's disease (AD) brains has identified numerous understudied proteins, including midkine (MDK), that are highly upregulated and correlated with Aβ since the early disease stage, but their roles in disease progression are not fully understood. Here we present that MDK attenuates Aβ assembly and influences amyloid formation in the 5xFAD amyloidosis mouse model. MDK protein mitigates fibril formation of both Aβ40 and Aβ42 peptides in Thioflavin T fluorescence assay, circular dichroism, negative stain electron microscopy, and NMR analysis. Knockout of Mdkgene in 5xFAD increases amyloid formation and microglial activation. Further comprehensive mass spectrometry-based profiling of whole proteome and aggregated proteome in these mouse models indicates significant accumulation of Aβ and Aβ-correlated proteins, along with microglial components. Thus, our structural and mouse model studies reveal a protective role of MDK in counteracting amyloid pathology in Alzheimer's disease.

A DNA-based molecular clamp for probing protein interactions and structure under force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA based device in which the transition between single-stranded and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Xinyang flavivirus, from Haemaphysalis flava ticks in Henan Province, China, defines a basal, likely tick-only Orthoflavivirus clade.

Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75 % in adult females and 15.19 % in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.

Detection of macropodid alphaherpesvirus 2 infection and lesions in sudden death of a captive Virginia opossum and a water opossum.

Macropodid alphaherpesvirus 2 (MaAHV2) is best described in macropods and has been implicated in outbreaks among captive marsupial populations in Australia. Natural disease caused by herpesviruses has not been reported previously in opossum species, to our knowledge. One Virginia opossum (Didelphis virginiana) and 1 water opossum (Chironectes minimus) were submitted for postmortem examination from a zoo that housed 6 opossums, all of which died within several weeks. Red kangaroos (Macropus rufus) and red-necked wallabies (Macropus rufogriseus) were also present at the facility. Liver samples from both opossums were submitted for transmission electron microscopy and whole-genome sequencing. Microscopically, both opossums had multifocal necrosis in the liver and lung, with intranuclear inclusion bodies within hepatocytes and pneumocytes. Another significant finding in the Virginia opossum was sepsis, with isolation of Streptococcus didelphis from various organs. Ultrastructural analysis of formalin-fixed liver tissue identified herpesviral replication complexes in both opossums; negative-stain electron microscopy of unfixed liver tissue repeatedly yielded a negative result. The herpesvirus had >99% nucleotide identity with MaAHV2. These 2 cases indicate that both opossum species are susceptible to MaAHV2 infection, and the outbreak has implications for mixed-species facilities that house macropods.