On the Relationship between MMA Levels in Blood Products and Donor Sex, Age, and Donation Frequency.

As the aging process accelerates, the age structure of blood donors turns to older and even aged groups. Methylmalonic acid (MMA), a byproduct of propionate metabolism, may be upregulated in the serum of older adults. As a mediator of chronic disease and tumor progression, the MMA content in blood products has become the focus of research. Absolute concentrations of MMA in blood products were determined based on the liquid chromatography-tandem mass spectrometry, so as to analyze how they were affected by donors' age, sex, and frequency of blood donation. The MMA content in leukocyte-depleted suspended red blood cell (lds-RBC) was significantly higher than that in fresh plasma (p<0.0001). The MMA content among five age groups showed no difference in either fresh plasma or lds-RBCs. The MMA content in fresh plasma was similar in the parameters of the sex, whereas that in lds-RBCs was higher in males than that in females (p=0.035). There were no significant differences in MMA content when it comes to different frequencies of blood donors in either fresh plasma or lds-RBCs. Additionally, there was no significant difference or clear trend in the rate of elevated plasma MMA levels among different sexes, age groups, and blood donation frequency groups. MMA in the blood products from donors in China does not compromise the safety of blood transfusions for cancer patients. Nevertheless, there is a need to focus on MMA levels in Chinese and to develop race-specific and age-specific normal reference ranges.

Protocol to detect infectious SARS-CoV-2 at low levels using in situ hybridization techniques.

Low and persistent levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA/protein/virus can be detected in clinical samples months after infection, possibly related to the emergence of SARS-CoV-2 variants or development of long coronavirus disease. Here, we present a protocol to detect low levels of viral RNA together with protein using flow cytometry and microscopy. We describe steps for cell infection with SARS-CoV-2 and quantification by fluorescence in situ hybridization-flow cytometry. We then detail procedures for visualization using immunolabeling and RNAscope. This approach is directly applicable to clinical samples. For complete details on the use and execution of this protocol, please refer to Zhu et al. (2022).1.

TRPV1 activation in human Langerhans cells and T cells inhibits mucosal HIV-1 infection via CGRP-dependent and independent mechanisms.

Upon its mucosal transmission, HIV type 1 (HIV-1) rapidly targets genital antigen-presenting Langerhans cells (LCs), which subsequently transfer infectious virus to CD4+ T cells. We previously described an inhibitory neuroimmune cross talk, whereby calcitonin gene-related peptide (CGRP), a neuropeptide secreted by peripheral pain-sensing nociceptor neurons innervating all mucosal epithelia and associating with LCs, strongly inhibits HIV-1 transfer. As nociceptors secret CGRP following the activation of their Ca2+ ion channel transient receptor potential vanilloid 1 (TRPV1), and as we reported that LCs secret low levels of CGRP, we investigated whether LCs express functional TRPV1. We found that human LCs expressed mRNA and protein of TRPV1, which was functional and induced Ca2+ influx following activation with TRPV1 agonists, including capsaicin (CP). The treatment of LCs with TRPV1 agonists also increased CGRP secretion, reaching its anti-HIV-1 inhibitory concentrations. Accordingly, CP pretreatment significantly inhibited LCs-mediated HIV-1 transfer to CD4+ T cells, which was abrogated by both TRPV1 and CGRP receptor antagonists. Like CGRP, CP-induced inhibition of HIV-1 transfer was mediated via increased CCL3 secretion and HIV-1 degradation. CP also inhibited direct CD4+ T cells HIV-1 infection, but in CGRP-independent manners. Finally, pretreatment of inner foreskin tissue explants with CP markedly increased CGRP and CCL3 secretion, and upon subsequent polarized exposure to HIV-1, inhibited an increase in LC-T cell conjugate formation and consequently T cell infection. Our results reveal that TRPV1 activation in human LCs and CD4+ T cells inhibits mucosal HIV-1 infection, via CGRP-dependent/independent mechanisms. Formulations containing TRPV1 agonists, already approved for pain relief, could hence be useful against HIV-1.

S100A8-mediated metabolic adaptation controls HIV-1 persistence in macrophages in vivo.

HIV-1 eradication is hindered by viral persistence in cell reservoirs, established not only in circulatory CD4+T-cells but also in tissue-resident macrophages. The nature of macrophage reservoirs and mechanisms of persistence despite combined anti-retroviral therapy (cART) remain unclear. Using genital mucosa from cART-suppressed HIV-1-infected individuals, we evaluated the implication of macrophage immunometabolic pathways in HIV-1 persistence. We demonstrate that ex vivo, macrophage tissue reservoirs contain transcriptionally active HIV-1 and viral particles accumulated in virus-containing compartments, and harbor an inflammatory IL-1R+S100A8+MMP7+M4-phenotype prone to glycolysis. Reactivation of infectious virus production and release from these reservoirs in vitro are induced by the alarmin S100A8, an endogenous factor produced by M4-macrophages and implicated in "sterile" inflammation. This process metabolically depends on glycolysis. Altogether, inflammatory M4-macrophages form a major tissue reservoir of replication-competent HIV-1, which reactivate viral production upon autocrine/paracrine S100A8-mediated glycolytic stimulation. This HIV-1 persistence pathway needs to be targeted in future HIV eradication strategies.

Persistent but dysfunctional mucosal SARS-CoV-2-specific IgA and low lung IL-1ß associate with COVID-19 fatal outcome: A cross-sectional analysis.

The role of the mucosal pulmonary antibody response in coronavirus disease 2019 (COVID-19) outcome remains unclear. Here, we found that in bronchoalveolar lavage (BAL) samples from 48 patients with severe COVID-19-infected with the ancestral Wuhan virus, mucosal IgG and IgA specific for S1, receptor-binding domain (RBD), S2, and nucleocapsid protein (NP) emerged in BAL containing viruses early in infection and persist after virus elimination, with more IgA than IgG for all antigens tested. Furthermore, spike-IgA and spike-IgG immune complexes were detected in BAL, especially when the lung virus has been cleared. BAL IgG and IgA recognized the four main RBD variants. BAL neutralizing titers were higher early in COVID-19 when virus replicates in the lung than later in infection after viral clearance. Patients with fatal COVID-19, in contrast to survivors, developed higher levels of mucosal spike-specific IgA than IgG but lost neutralizing activities over time and had reduced IL-1ß in the lung. Altogether, mucosal spike and NP-specific IgG and S1-specific IgA persisting after lung severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance and low pulmonary IL-1ß correlate with COVID-19 fatal outcome. Thus, mucosal SARS-CoV-2-specific antibodies may have adverse functions in addition to protective neutralization. Highlights: Mucosal pulmonary antibody response in COVID-19 outcome remains unclear. We show that in severe COVID-19 patients, mucosal pulmonary non-neutralizing SARS-CoV-2 IgA persit after viral clearance in the lung. Furthermore, low lung IL-1ß correlate with fatal COVID-19. Altogether, mucosal IgA may exert harmful functions beside protective neutralization.

Infection of lung megakaryocytes and platelets by SARS-CoV-2 anticipate fatal COVID-19.

SARS-CoV-2, although not being a circulatory virus, spread from the respiratory tract resulting in multiorgan failures and thrombotic complications, the hallmarks of fatal COVID-19. A convergent contributor could be platelets that beyond hemostatic functions can carry infectious viruses. Here, we profiled 52 patients with severe COVID-19 and demonstrated that circulating platelets of 19 out 20 non-survivor patients contain SARS-CoV-2 in robust correlation with fatal outcome. Platelets containing SARS-CoV-2 might originate from bone marrow and lung megakaryocytes (MKs), the platelet precursors, which were found infected by SARS-CoV-2 in COVID-19 autopsies. Accordingly, MKs undergoing shortened differentiation and expressing anti-viral IFITM1 and IFITM3 RNA as a sign of viral sensing were enriched in the circulation of deadly COVID-19. Infected MKs reach the lung concomitant with a specific MK-related cytokine storm rich in VEGF, PDGF and inflammatory molecules, anticipating fatal outcome. Lung macrophages capture SARS-CoV-2-containing platelets in vivo. The virus contained by platelets is infectious as capture of platelets carrying SARS-CoV-2 propagates infection to macrophages in vitro, in a process blocked by an anti-GPIIbIIIa drug. Altogether, platelets containing infectious SARS-CoV-2  alter COVID-19 pathogenesis and provide a powerful fatality marker. Clinical targeting of platelets might prevent viral spread, thrombus formation and exacerbated inflammation at once and increase survival in COVID-19.

Therapeutic potential of melatonin and melatonergic drugs on K18-hACE2 mice infected with SARS-CoV-2.

As the COVID-19 pandemic grows, several therapeutic candidates are being tested or undergoing clinical trials. Although prophylactic vaccination against SARS-CoV-2 infection has been shown to be effective, no definitive treatment exists to date in the event of infection. The rapid spread of infection by SARS-CoV-2 and its variants fully warrants the continued evaluation of drug treatments for COVID-19, especially in the context of repurposing of already available and safe drugs. Here, we explored the therapeutic potential of melatonin and melatonergic compounds in attenuating COVID-19 pathogenesis in mice expressing human ACE2 receptor (K18-hACE2), strongly susceptible to SARS-CoV-2 infection. Daily administration of melatonin, agomelatine, or ramelteon delays the occurrence of severe clinical outcome with improvement of survival, especially with high melatonin dose. Although no changes in most lung inflammatory cytokines are observed, treatment with melatonergic compounds limits the exacerbated local lung production of type I and type III interferons, which is likely associated with the observed improved symptoms in treated mice. The promising results from this preclinical study should encourage studies examining the benefits of repurposing melatonergic drugs to treat COVID-19 and related diseases in humans.

Viral Metagenome-Based Precision Surveillance of Pig Population at Large Scale Reveals Viromic Signatures of Sample Types and Influence of Farming Management on Pig Virome.

Pigs are a major meat source worldwide and a pillar of Chinese animal husbandry; hence, their health and safety are a prioritized concern of the national economy. Although pig viruses have been continuously investigated, the full extent of the pig virome has remained unknown and emerging viruses are still a major threat to the pig industry. Here, we report a comprehensive study to delineate the pig virome of 1,841 healthy weaned pigs from 45 commercial farms collected from 25 major pig-producing regions across China. A viromic sequence data set, named Pigs_VIRES, which matched 96,586 viral genes from at least 249 genera within 66 families and which almost tripled the number of previously published pig viromic genes, was established. The majority of the mammalian viruses were closely related to currently known ones. A comparison with previously published viromes of bovines, avians, and humans has revealed the distinct composition of Pigs_VIRES, which has provided characteristic viromic signatures of serum, pharyngeal, and anal samples that were significantly influenced by farming management and disease control measures. Taken together, Pigs_VIRES has revealed the most complete viromic data set of healthy pigs to date. The compiled data also provide useful guidance to pig viral disease control and prevention and the biosafety management of pig farms. Especially, the established viromic protocol has created a precision surveillance strategy to potentially innovate currently used surveillance methods of animal infectious diseases, particularly by making precision surveillance available to other animal species on a large scale or even during a nationwide surveillance campaign. IMPORTANCE Pigs are deeply involved in human lives; hence, their viruses are associated with public health. Here, we established the most comprehensive virome of healthy piglets to date, which provides a viromic baseline of weaned pigs for disease prevention and control, highlighting that longitudinal viromic monitoring is needed to better understand the dynamics of the virome in pig development and disease occurrence. The present study also shows how high standards of animal farm management with strict biosafety measures can significantly minimize the risk of introduction of pathogenic viruses into pig farms. Particularly, the viromic strategy established, i.e., high-throughput detection and analyses of various known and unknown pathogenic viruses in a single test at large scale, has completely innovated current surveillance measures in provision of timely and precise detection of all potentially existing pathogenic viruses and can be widely applied in other animal species.

HIV-Sheltering Platelets From Immunological Non-Responders Induce a Dysfunctional Glycolytic CD4+ T-Cell Profile.

Immunological non-responders (InRs) are HIV-infected individuals in whom the administration of combination antiretroviral therapy (cART), although successful in suppressing viral replication, cannot properly reconstitute patient circulating CD4+ T-cell number to immunocompetent levels. The causes for this immunological failure remain elusive, and no therapeutic strategy is available to restore a proper CD4+ T-cell immune response in these individuals. We have recently demonstrated that platelets harboring infectious HIV are a hallmark of InR, and we now report on a causal connection between HIV-containing platelets and T-cell dysfunctions. We show here that in vivo, platelet-T-cell conjugates are more frequent among CD4+ T cells in InRs displaying HIV-containing platelets (<350 CD4+ T cells/µl blood for >1 year) as compared with healthy donors or immunological responders (IRs; >350 CD4+ T cells/µl). This contact between platelet containing HIV and T cell in the conjugates is not infectious for CD4+ T cells, as coculture of platelets from InRs containing HIV with healthy donor CD4+ T cells fails to propagate infection to CD4+ T cells. In contrast, when macrophages are the target of platelets containing HIV from InRs, macrophages become infected. Differential transcriptomic analyses comparing InR and IR CD4+ T cells reveal an upregulation of genes involved in both aerobic and anaerobic glycolysis in CD4+ T cells from InR vs. IR individuals. Accordingly, InR platelets containing HIV induce a dysfunctional increase in glycolysis-mediated energy production in CD4+ T cells as compared with T cells cocultured with IR platelets devoid of virus. In contrast, macrophage metabolism is not affected by platelet contact. Altogether, this brief report demonstrates a direct causal link between presence of HIV in platelets and T-cell dysfunctions typical of InR, contributing to devise a platelet-targeted therapy for improving immune reconstitution in these individuals.

Platelets from HIV-infected individuals on antiretroviral drug therapy with poor CD4+ T cell recovery can harbor replication-competent HIV despite viral suppression.

In addition to hemostasis, human platelets have several immune functions and interact with infectious pathogens including HIV in vitro. Here, we report that platelets from HIV-infected individuals on combined antiretroviral drug therapy (ART) with low blood CD4+ T cell counts (<350 cells/µl) contained replication-competent HIV despite viral suppression. In vitro, human platelets harboring HIV propagated the virus to macrophages, a process that could be prevented with the biologic abciximab, an anti-integrin αIIb/ß3 Fab. Furthermore, in our cohort, 88% of HIV-infected individuals on ART with viral suppression and with platelets containing HIV were poor immunological responders with CD4+ T cell counts remaining below <350 cells/µl for more than one year. Our study suggests that platelets may be transient carriers of HIV and may provide an alternative pathway for HIV dissemination in HIV-infected individuals on ART with viral suppression and poor CD4+ T cell recovery.

Extensive Genetic Diversity of Polyomaviruses in Sympatric Bat Communities: Host Switching versus Coevolution.

Polyomaviruses (PyVs) are small DNA viruses carried by diverse vertebrates. The evolutionary relationships of viruses and hosts remain largely unclear due to very limited surveillance in sympatric communities. In order to investigate whether PyVs can transmit among different mammalian species and to identify host-switching events in the field, we conducted a systematic study of a large collection of bats (n = 1,083) from 29 sympatric communities across China which contained multiple species with frequent contact. PyVs were detected in 21 bat communities, with 192 PyVs identified in 186 bats from 15 species within 6 families representing at least 28 newly described PyVs. Surveillance results and phylogenetic analyses surprisingly revealed three interfamily PyV host-switching events in these sympatric bat communities: two distinct PyVs were identified in two bat species in restricted geographical locations, while another PyV clustered phylogenetically with PyVs carried by bats from a different host family. Virus-host relationships of all discovered PyVs were also evaluated, and no additional host-switching events were found. PyVs were identified in different horseshoe bat species in sympatric communities without observation of host-switching events, showed high genomic identities, and clustered with each other. This suggested that even for PyVs with high genomic identities in closely related host species, the potential for host switching is low. In summary, our findings revealed that PyV host switching in sympatric bat communities can occur but is limited and that host switching of bat-borne PyVs is relatively rare on the predominantly evolutionary background of codivergence with their hosts.IMPORTANCE Since the discovery of murine polyomavirus in the 1950s, polyomaviruses (PyVs) have been considered highly host restricted in mammals. Sympatric bat communities commonly contain several different bat species in an ecological niche facilitating viral transmission, and they therefore represent a model to identify host-switching events of PyVs. In this study, we screened PyVs in a large number of bats in sympatric communities from diverse habitats across China. We provide evidence that cross-species bat-borne PyV transmission exists, though is limited, and that host-switching events appear relatively rare during the evolutionary history of these viruses. PyVs with close genomic identities were also identified in different bat species without host-switching events. Based on these findings, we propose an evolutionary scheme for bat-borne PyVs in which limited host-switching events occur on the background of codivergence and lineage duplication, generating the viral genetic diversity in bats.

Molecular characterization of a novel bat-associated circovirus with a poly-T tract in the 3' intergenic region.

The family Circoviridae comprises a large group of small circular single-stranded DNA viruses with several members causing severe pig and poultry diseases. In recent years the number of new viruses within the family has had an explosive increase showing a high level of genetic diversity and a broad host range. In this report we describe two more circoviruses identified from bats in Yunnan and Heilongjiang provinces in China. Full genome sequencing has revealed that these bat associated circoviruses (bat ACV) should be classified as new species within the genus Circovirus based on the demarcation criteria of the International Committee on the Taxonomy of Viruses (ICTV). The most striking result is the novel finding of a 21-28 nt polythymidine (poly-T) tract in the 3' terminal intergenic region of bat ACV isolates from Heilongjiang province. To understand its role in viral replication, a wild type bat ACV and a mutated version with the entire poly-T deleted were rescued through construction of infectious clones. Replication comparison in vitro showed that the poly-T is not essential for viral replication. Identification of additional bat ACV isolates and study of their biological characteristics will be the main task in future to understand the potential roles of bats in transmission of circoviruses to terrestrial mammals and humans.