Comparative Performance in the Detection of Four Coronavirus Genera from Human, Animal, and Environmental Specimens.

Emerging coronaviruses (CoVs) are understood to cause critical human and domestic animal diseases; the spillover from wildlife reservoirs can result in mild and severe respiratory illness in humans and domestic animals and can spread more readily in these naïve hosts. A low-cost CoV molecular method that can detect a variety of CoVs from humans, animals, and environmental specimens is an initial step to ensure the early identification of known and new viruses. We examine a collection of 50 human, 46 wastewater, 28 bat, and 17 avian archived specimens using 3 published pan-CoV PCR assays called Q-, W-, and X-CoV PCR, to compare the performance of each assay against four CoV genera. X-CoV PCR can detect all four CoV genera, but Q- and W-CoV PCR failed to detect δ-CoV. In total, 21 (42.0%), 9 (18.0%), and 21 (42.0%) of 50 human specimens and 30 (65.22%), 6 (13.04%), and 27 (58.70%) of 46 wastewater specimens were detected using Q-, W-, and X-CoV PCR assays, respectively. The X-CoV PCR assay has a comparable sensitivity to Q-CoV PCR in bat CoV detection. Combining Q- and X-CoV PCR assays can increase sensitivity and avoid false negative results in the early detection of novel CoVs.

An unusual diarrheal outbreak in the community in Eastern Thailand caused by Norovirus GII.3[P25].

BACKGROUND: Sentinel laboratory surveillance for diarrheal disease determined norovirus to be the most common cause of non-bacterial gastroenteritis in people during the COVID-19 pandemic in Thailand. An increase in patients presenting with diarrhea and vomiting in hospitals across Chanthaburi province between December 2021 and January 2022 led to the need for the identification of viral pathogens that may be responsible for the outbreak. METHODS: Fecal samples (rectal swabs or stool) from 93 patients, of which 65 patients were collected during the December 2021 to January 2022 outbreak, were collected and screened for viral infection by real-time RT-PCR. Positive samples for norovirus GII were then genotyped by targeted amplification and sequencing of partial polymerase and capsid genes. Full genome sequencing was performed from the predominant strain, GII.3[P25]. RESULTS: Norovirus was the most common virus detected in human fecal samples in this study. 39 of 65 outbreak samples (60%) and 3 of 28 (10%) non-outbreak samples were positive for norovirus genogroup II. One was positive for rotavirus, and one indicated co-infection with rotavirus and norovirus genogroups I and II. Nucleotide sequences of VP1 and RdRp gene were successfully obtained from 28 of 39 positive norovirus GII and used for dual-typing; 25/28 (89.3%) were GII.3, and 24/28 (85.7) were GII.P25, respectively. Norovirus GII.3[P25] was the predominant strain responsible for this outbreak. The full genome sequence of norovirus GII.3[P25] from our study is the first reported in Thailand and has 98.62% and 98.57% similarity to norovirus found in China in 2021 and the USA in 2022, respectively. We further demonstrate the presence of multiple co-circulating norovirus genotypes, including GII.21[P21], GII.17[P17], GII.3[P12] and GII.4[P31] in our study. CONCLUSIONS: An unusual diarrhea outbreak was found in December 2021 in eastern Thailand. Norovirus strain GII.3[P25] was the cause of the outbreak and was first detected in Thailand. The positive rate during GII.3[P25] outbreak was six times higher than sporadic cases (GII.4), and, atypically, adults were the primary infected population rather than children.

Antibody targeting of conserved sites of vulnerability on the SARS-CoV-2 spike receptor-binding domain.

Given the continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs), immunotherapeutics that target conserved epitopes on the spike (S) glycoprotein have therapeutic advantages. Here, we report the crystal structure of the SARS-CoV-2 S receptor-binding domain (RBD) at 1.95 Å and describe flexibility and distinct conformations of the angiotensin-converting enzyme 2 (ACE2)-binding site. We identify a set of SARS-CoV-2-reactive monoclonal antibodies (mAbs) with broad RBD cross-reactivity including SARS-CoV-2 Omicron subvariants, SARS-CoV-1, and other sarbecoviruses and determine the crystal structures of mAb-RBD complexes with Ab246 and CR3022 mAbs targeting the class IV site, WRAIR-2134, which binds the recently designated class V epitope, and WRAIR-2123, the class I ACE2-binding site. The broad reactivity of class IV and V mAbs to conserved regions of SARS-CoV-2 VoCs and other sarbecovirus provides a framework for long-term immunotherapeutic development strategies.

Recombinant Soluble Henipavirus Glycoprotein Preparation.

Henipaviruses possess two envelope glycoproteins, the attachment (G) and the fusion (F) proteins that mediate cellular entry and are the major targets of virus-neutralizing antibody responses. Recombinant expression technologies have been used to produce soluble G and F proteins (sG and sF) that retain native-like oligomeric conformations and epitopes, which are advantageous for the development and characterization of vaccines and antiviral antibody therapeutics. In addition to Hendra virus and Nipah virus tetrameric sG and trimeric sF production, we also describe the expression and purification of Cedar virus tetrameric sG and Ghana virus trimeric sF glycoproteins. These henipavirus glycoproteins were also used as immunizing antigens to generate monoclonal antibodies, and binding was demonstrated with a pan-henipavirus multiplex microsphere immunoassay.

Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity.

Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.

Bma-LAD-2, an Intestinal Cell Adhesion Protein, as a Potential Therapeutic Target for Lymphatic Filariasis.

Lymphatic filariasis is a debilitating disease that afflicts over 70 million people worldwide. It is caused by the parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. Despite substantial success, efforts to eliminate LF will likely require more time and resources than predicted. Identifying new drug and vaccine targets in adult filariae could help elimination efforts. This study's aim was to evaluate intestinal proteins in adult Brugia malayi worms as possible therapeutic targets. Using short interfering RNA (siRNA), we successfully targeted four candidate gene transcripts: Bma-Serpin, Bma-ShTK, Bma-Reprolysin, and Bma-LAD-2. Of those, Bma-LAD-2, an immunoglobulin superfamily cell adhesion molecule (IgSF CAM), was determined to be essential for adult worm survival. We observed a 70.42% knockdown in Bma-LAD-2 transcript levels 1 day post-siRNA incubation and an 87.02% reduction in protein expression 2 days post-siRNA incubation. This inhibition of Bma-LAD-2 expression resulted in an 80% decrease in worm motility over 6 days, a 93.43% reduction in microfilaria release (Mf) by day 6 post-siRNA incubation, and a dramatic decrease in (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Transmission electron microscopy revealed the loss of microvilli and unraveling of mitochondrial cristae in the intestinal epithelium of Bma-LAD-2 siRNA-treated worms. Strikingly, Bma-LAD-2 siRNA-treated worms exhibited an almost complete loss of pseudocoelomic fluid. A luciferase immunoprecipitation system assay did not detect anti-Bma-LAD-2 IgE in the serum of 30 LF patients, indicating that LF exposure does not result in IgE sensitization to this antigen. These results indicate that Bma-LAD-2 is an essential protein for adult Brugia malayi and may be an effective therapeutic target. IMPORTANCE Brugia malayi is a parasitic nematode that can cause lymphatic filariasis, a debilitating disease prevalent in tropical and subtropical countries. Significant progress has been made toward eliminating the disease. However, complete eradication may require new therapeutics such as drugs or a vaccine that kill adult filariae. In this study, we identified an immunoglobulin superfamily cell adhesion molecule (Bma-LAD-2) as a potential drug and vaccine candidate. When we knocked down Bma-LAD-2 expression, we observed a decrease in worm motility, fecundity, and metabolism. We also visualized the loss of microvilli, destruction of the mitochondria in the intestinal epithelium, and loss of pseudocoelomic fluid contents after Bma-LAD-2 siRNA treatment. Finally, we demonstrated that serum from filaria-infected patients does not contain preexisting IgE to Bma-LAD-2, which indicates that this antigen would be safe to administer as a vaccine in populations where the disease is endemic.

Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Durability of Antibody Response and Frequency of SARS-CoV-2 Infection 6 Months after COVID-19 Vaccination in Healthcare Workers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies decay but persist 6 months postvaccination; lower levels of neutralizing titers persist against Delta than wild-type virus. Of 227 vaccinated healthcare workers tested, only 2 experienced outpatient symptomatic breakthrough infections, despite 59/227 exhibiting serologic evidence of SARS-CoV-2 infection, defined as presence of nucleocapsid protein antibodies.

Antigen-based multiplex strategies to discriminate SARS-CoV-2 natural and vaccine induced immunity from seasonal human coronavirus humoral responses.

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 sero-surveillance. With the rollout of SARS-CoV-2 vaccines, such assays must be able to distinguish vaccine from natural immunity to SARS-CoV-2 and related human coronaviruses. Here, we developed and implemented multiplex microsphere-based immunoassay strategies for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2 and the endemic seasonal human coronaviruses (HCoV), enabling high throughout measurement of pre-existing cross-reactive antibodies. We varied SARS-CoV-2 antigen compositions within the multiplex assay, allowing direct comparisons of the effects of spike protein, receptor-binding domain protein (RBD) and nucleocapsid protein (NP) based SARS-CoV-2 antibody detection. Multiplex immunoassay performance characteristics are antigen-dependent, and sensitivities and specificities range 92-99% and 94-100%, respectively, for human subject samples collected as early as 7-10 days from symptom onset. SARS-CoV-2 spike and RBD had a strong correlative relationship for the detection of IgG. Correlation between detectable IgG reactive with spike and NP also had strong relationship, however, several PCR-positive and spike IgG-positive serum samples were NP IgG-negative. This spike and NP multiplex immunoassay has the potential to be useful for differentiation between vaccination and natural infection induced antibody responses. We also assessed the induction of de novo SARS-CoV-2 IgG cross reactions with SARS-CoV and MERS-CoV spike proteins. Furthermore, multiplex immunoassays that incorporate spike proteins of SARS-CoV-2 and HCoVs will permit investigations into the influence of HCoV antibodies on COVID-19 clinical outcomes and SARS-CoV-2 antibody durability.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and antibody cross reactions.

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we developed and applied a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human betacoronaviruses, HCoV-HKU1 and HCoV-OC43, that enables measurement of off-target pre-existing cross-reactive antibodies. The MMIA performances characteristics are: 98% sensitive and 100% specific for human subject samples collected as early as 10 days from symptom onset. The MMIA permitted the simultaneous identification of SARS-CoV-2 seroconversion and the induction of SARS-CoV-2 IgG antibody cross reactions to SARS-CoV-1 and MERS-CoV. Further, synchronous increases of HCoV-OC43 IgG antibody levels was detected with SARS-CoV-2 seroconversion in a subset of subjects for whom early infection sera were available prior to their SARS-CoV-2 seroconversion, suggestive of an HCoV-OC43 memory response triggered by SARS-CoV-2 infection.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity.

With growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. military hospitals. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. Requiring only 1.25 µL of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.

A Cryptic Site of Vulnerability on the Receptor Binding Domain of the SARS-CoV-2 Spike Glycoprotein.

SARS-CoV-2 is a zoonotic virus that has caused a pandemic of severe respiratory disease-COVID-19-within several months of its initial identification. Comparable to the first SARS-CoV, this novel coronavirus's surface Spike (S) glycoprotein mediates cell entry via the human ACE-2 receptor, and, thus, is the principal target for the development of vaccines and immunotherapeutics. Molecular information on the SARS-CoV-2 S glycoprotein remains limited. Here we report the crystal structure of the SARS-CoV-2 S receptor-binding-domain (RBD) at a the highest resolution to date, of 1.95 Å. We identified a set of SARS-reactive monoclonal antibodies with cross-reactivity to SARS-CoV-2 RBD and other betacoronavirus S glycoproteins. One of these antibodies, CR3022, was previously shown to synergize with antibodies that target the ACE-2 binding site on the SARS-CoV RBD and reduce viral escape capacity. We determined the structure of CR3022, in complex with the SARS-CoV-2 RBD, and defined a broadly reactive epitope that is highly conserved across betacoronaviruses. This epitope is inaccessible in the "closed" prefusion S structure, but is accessible in "open" conformations. This first-ever resolution of a human antibody in complex with SARS-CoV-2 and the broad reactivity of this set of antibodies to a conserved betacoronavirus epitope will allow antigenic assessment of vaccine candidates, and provide a framework for accelerated vaccine, immunotherapeutic and diagnostic strategies against SARS-CoV-2 and related betacoronaviruses.

Serological Evidence for Henipa-like and Filo-like Viruses in Trinidad Bats.

Bat-borne zoonotic pathogens belonging to the family Paramxyoviridae, including Nipah and Hendra viruses, and the family Filoviridae, including Ebola and Marburg viruses, can cause severe disease and high mortality rates on spillover into human populations. Surveillance efforts for henipaviruses and filoviruses have been largely restricted to the Old World; however, recent studies suggest a potentially broader distribution for henipaviruses and filoviruses than previously recognized. In the current study, we screened for henipaviruses and filoviruses in New World bats collected across 4 locations in Trinidad near the coast of Venezuela. Bat tissue samples were screened using previously established reverse-transcription polymerase chain reaction assays. Serum were screened using a multiplex immunoassay to detect antibodies reactive with the envelope glycoprotein of viruses in the genus Henipavirus and the family Filoviridae. Serum samples were also screened by means of enzyme-linked immunosorbent assay for antibodies reactive with Nipah G and F glycoproteins. Of 84 serum samples, 28 were reactive with ≥1 henipavirus glycoprotein by ≥1 serological method, and 6 serum samples were reactive against ≥1 filovirus glycoproteins. These data provide evidence of potential circulation of viruses related to the henipaviruses and filoviruses in New World bats.

Structural and functional analyses reveal promiscuous and species specific use of ephrin receptors by Cedar virus.

Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only ∼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.

Intestinal UDP-glucuronosyltransferase as a potential target for the treatment and prevention of lymphatic filariasis.

Lymphatic filariasis (LF), a morbid disease caused by the tissue-invasive nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, affects millions of people worldwide. Global eradication efforts have significantly reduced worldwide prevalence, but complete elimination has been hampered by limitations of current anti-filarial drugs and the lack of a vaccine. The goal of this study was to evaluate B. malayi intestinal UDP-glucuronosyltransferase (Bm-UGT) as a potential therapeutic target. To evaluate whether Bm-UGT is essential for adult filarial worms, we inhibited its expression using siRNA. This resulted in a 75% knockdown of Bm-ugt mRNA for 6 days and almost complete suppression of detectable Bm-UGT by immunoblot. Reduction in Bm-UGT expression resulted in decreased worm motility for 6 days, 70% reduction in microfilaria release from adult worms, and significant reduction in adult worm metabolism as detected by MTT assays. Because prior allergic-sensitization to a filarial antigen would be a contraindication for its use as a vaccine candidate, we tested plasma from infected and endemic normal populations for Bm-UGT-specific IgE using a luciferase immunoprecipitation assay. All samples (n = 35) tested negative. We then tested two commercially available medicines known to be broad inhibitors of UGTs, sulfinpyrazone and probenecid, for in vitro activity against B. malayi. There were marked macrofilaricidal effects at concentrations achievable in humans and very little effect on microfilariae. In addition, we observed that probenecid and sulfinpyrazone exhibit a synergistic macrofilaricidal effect when used in combination with albendazole. The results of this study demonstrate that Bm-UGT is an essential protein for adult worm survival. Lack of prior IgE sensitization in infected and endemic populations suggest it may be a feasible vaccine candidate. The finding that sulfinpyrazone and probenecid have in vitro effects against adult B. malayi worms suggests that these medications have promise as potential macrofilaricides in humans.

Enhanced Autophagy Contributes to Reduced Viral Infection in Black Flying Fox Cells.

Bats are increasingly implicated as hosts of highly pathogenic viruses. The underlying virus⁻host interactions and cellular mechanisms that promote co-existence remain ill-defined, but physiological traits such as flight and longevity are proposed to drive these adaptations. Autophagy is a cellular homeostatic process that regulates ageing, metabolism, and intrinsic immune defense. We quantified basal and stimulated autophagic responses in black flying fox cells, and demonstrated that although black flying fox cells are susceptible to Australian bat lyssavirus (ABLV) infection, viral replication is dampened in these bat cells. Black flying fox cells tolerated prolonged ABLV infection with less cell death relative to comparable human cells, suggesting post-entry mechanisms interference with virus replication. An elevated basal autophagic level was observed and autophagy was induced in response to high virus doses. Pharmacological stimulation of the autophagy pathway reduced virus replication, indicating autophagy acts as an anti-viral mechanism. Enhancement of basal and virus-induced autophagy in bat cells connects related reports that long-lived species possess homeostatic processes that dampen oxidative stress and macromolecule damage. Exemplifying the potential that evolved cellular homeostatic adaptations like autophagy may secondarily act as anti-viral mechanisms, enabling bats to serve as natural hosts to an assortment of pathogenic viruses. Furthermore, our data suggest autophagy-inducing drugs may provide a novel therapeutic strategy for combating lyssavirus infection.

Serologic Evidence of Fruit Bat Exposure to Filoviruses, Singapore, 2011-2016.

To determine whether fruit bats in Singapore have been exposed to filoviruses, we screened 409 serum samples from bats of 3 species by using a multiplex assay that detects antibodies against filoviruses. Positive samples reacted with glycoproteins from Bundibugyo, Ebola, and Sudan viruses, indicating filovirus circulation among bats in Southeast Asia.