Blockade of Autocrine CCL5 Responses Inhibits Zika Virus Persistence and Spread in Human Brain Microvascular Endothelial Cells

Our findings demonstrate that CCL5 is required for ZIKV to persistently infect human brain ECs that normally protect neuronal compartments. We demonstrate that ZIKV-elicited CCL5 secretion directs autocrine hBMEC activation of ERK1/2 survival pathways via CCR3/CCR5, and inhibiting CCL5/CCR3/CCR5 responses prevented ZIKV persistence and spread.

Biofilms and coronaviruses reservoirs: a perspective review

Bats are a key reservoir of coronaviruses (CoVs), including the agent of the severe acute respiratory syndrome, SARS-CoV-2, responsible for the recent deadly viral pneumonia pandemic. However, understanding how bats can harbor several microorganisms without developing illnesses is still a matter under discussion. Viruses and other pathogens are often studied as stand-alone even though it is known that, in nature, they mostly live in multi-species associations called biofilms - both externally and within the host. Microorganisms in biofilms are enclosed by an extracellular matrix that confers protection and improves survival. Previous studies have shown that viruses can secondarily colonize preexisting biofilms, and viral biofilms have also been already described. In this review, we raise the perspective that CoVs can persistently infect bats due to occurrence in biofilm structures. This phenomenon potentially provides an optimal environment for non-pathogenic and well-adapted viruses to interact with the host, as well as for viral recombination. Biofilms can also enhance virion viability in extracellular environments, such as in fomites and aquatic sediments, allowing viral persistence and dissemination. Moreover, understanding CoVs biofilm lifestyle in reservoirs might contribute to explain several burning questions that remain unanswered including persistence and transmissibility by highly pathogenic emerging CoVs.

NS5 Sumoylation Directs Nuclear Responses That Permit Zika Virus To Persistently Infect Human Brain Microvascular Endothelial Cells

ABSTRACT Zika virus (ZIKV) is cytopathic to neurons and persistently infects brain microvascular endothelial cells (hBMECs), which normally restrict viral access to neurons. Despite replicating in the cytoplasm, ZIKV and Dengue virus (DENV) polymerases, NS5 proteins, are predominantly trafficked to the nucleus. We found that a SUMO interaction motif in ZIKV and DENV NS5 proteins directs nuclear localization. However, ZIKV NS5 formed discrete punctate nuclear bodies (NBs), while DENV NS5 was uniformly dispersed in the nucleoplasm. Yet, mutating one DENV NS5 SUMO site (K546R) localized the NS5 mutant to discrete NBs, and NBs formed by the ZIKV NS5 SUMO mutant (K252R) were restructured into discrete protein complexes. In hBMECs, NBs formed by STAT2 and promyelocytic leukemia (PML) protein are present constitutively and enhance innate immunity. During ZIKV infection or NS5 expression, we found that ZIKV NS5 evicts PML from STAT2 NBs, forming NS5/STAT2 NBs that dramatically reduce PML expression in hBMECs and inhibit the transcription of interferon-stimulated genes (ISG). Expressing the ZIKV NS5 SUMO site mutant (K252R) resulted in NS5/STAT2/PML NBs that failed to degrade PML, reduce STAT2 expression, or inhibit ISG induction. Additionally, the K252 SUMOylation site and NS5 nuclear localization were required for ZIKV NS5 to regulate hBMEC cell cycle transcriptional responses. Our data reveal NS5 SUMO motifs as novel NB coordinating factors that distinguish flavivirus NS5 proteins. These findings establish SUMOylation of ZIKV NS5 as critical in the regulation of antiviral ISG and cell cycle responses that permit ZIKV to persistently infect hBMECs. IMPORTANCE ZIKV is a unique neurovirulent flavivirus that persistently infects human brain microvascular endothelial cells (hBMECs), the primary barrier that restricts viral access to neuronal compartments. Here, we demonstrate that flavivirus-specific SIM and SUMO sites determine the assembly of NS5 proteins into discrete nuclear bodies (NBs). We found that NS5 SIM sites are required for NS5 nuclear localization and that SUMO sites regulate NS5 NB complex constituents, assembly, and function. We reveal that ZIKV NS5 SUMO sites direct NS5 binding to STAT2, disrupt the formation of antiviral PML-STAT2 NBs, and direct PML degradation. ZIKV NS5 SUMO sites also transcriptionally regulate cell cycle and ISG responses that permit ZIKV to persistently infect hBMECs. Our findings demonstrate the function of SUMO sites in ZIKV NS5 NB formation and their importance in regulating nuclear responses that permit ZIKV to persistently infect hBMECs and thereby gain access to neurons.

Adventitious viruses persistently infect three commonly used mosquito cell lines

Mosquito cell lines were first established in the 1960’s and have been used extensively in research to isolate and propagate arthropod-borne (arbo-) viruses, study the invertebrate immune system, and understand virus-vector interactions. Despite their utility as anin vitrotool, these cell lines are poorly defined and may harbor insect-specific viruses that could impact experimental results. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells fromAedes albopictus, Aag2 cells fromAedes aegypti, and Hsu cells fromCulex quinquefasciatus, for the presence of adventitious viruses. All four cell lines stained positive for double-stranded RNA by immunofluorescence, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. Sequences from viruses in the familiesBirnaviridae,Bunyaviridae, Flaviviridae,andRhabdoviridaewere abundant in the mosquito cell lines. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems.

Pseudomonas aeruginosa and Klebsiella pneumoniae Adaptation to Innate Immune Clearance Mechanisms in the Lung

Many different species of gram-negative bacteria are associated with infection in the lung, causing exacerbations of chronic obstructive pulmonary disease, cystic fibrosis (CF), and ventilator-associated pneumonias. These airway pathogens must adapt to common host clearance mechanisms that include killing by antimicrobial peptides, antibiotics, oxidative stress, and phagocytosis by leukocytes. Bacterial adaptation to the host is often evident phenotypically, with increased extracellular polysaccharide production characteristic of some biofilm-associated organisms. Given the relatively limited repertoire of bacterial strategies to elude airway defenses, it seems likely that organisms sharing the same ecological niche might also share common strategies to persistently infect the lung. In this review, we will highlight some of the major factors responsible for the adaptation of Pseudomonas aeruginosa to the lung, addressing how growth in biofilms enables persistent infection, relevant to, but not limited to, the pathogenesis of infection in CF. In contrast, we will discuss how carbapenem-resistant Klebsiella pneumoniae evade immune clearance, an organism often associated with ventilator-associated pneumonia and health-care-acquired pneumonias, but not a typical pathogen in CF.

Lymantria dispar iflavirus 1 (LdIV1), a new model to study iflaviral persistence in lepidopterans

The cell line IPLB-LD-652Y, derived from the gypsy moth (Lymantria dispar L.), is routinely used to study interactions between viruses and insect hosts. Here we report the full genome sequence and biological characteristics of a small RNA virus, designated Lymantria dispar iflavirus 1 (LdIV1), that was discovered to persistently infect IPLB-LD-652Y. LdIV1 belongs to the genus Iflavirus. LdIV1 formed icosahedral particles of approx. 30 nm in diameter and contained a 10 044 nt polyadenylated, positive-sense RNA genome encoding a predicted polyprotein of 2980 aa. LdIV1 was induced by a viral suppressor of RNA silencing, suggesting that acute infection is restricted by RNA interference (RNAi). We detected LdIV1 in all tested tissues of gypsy-moth larvae and adults, but the virus was absent from other L. dispar-derived cell lines. We confirmed LdIV1 infectivity in two of these cell lines (IPLB-LD-652 and IPLB-LdFB). Our results provide a novel system to explore persistent infections in lepidopterans and a new model for the study of iflaviruses, a rapidly expanding group of viruses, many of which covertly infect their hosts.

The Borrelia burgdorferi outer-surface protein ErpX binds mammalian laminin

The Lyme disease spirochaete, Borrelia burgdorferi, can invade and persistently infect its hosts' connective tissues. We now demonstrate that B. burgdorferi adheres to the extracellular matrix component laminin. The surface-exposed outer-membrane protein ErpX was identified as having affinity for laminin, and is the first laminin-binding protein to be identified in a Lyme disease spirochaete. The adhesive domain of ErpX was shown to be contained within a small, unstructured hydrophilic segment at the protein's centre. The sequence of that domain is distinct from any previously identified bacterial laminin adhesin, suggesting a unique mode of laminin binding.

Fitness Variation of Borrelia burgdorferi Sensu Stricto Strains in Mice

ABSTRACT Lyme borreliosis in North America is caused by the tick-borne spirochete Borrelia burgdorferi, a zoonotic bacterium that is able to persistently infect a wide range of vertebrate species. Given the pronounced strain structure of B. burgdorferi in the northeastern United States, we asked whether the fitness of the different genotypes varies among susceptible vertebrate hosts. The transmission dynamics of two genetically divergent human isolates of B. burgdorferi, BL206 and B348, were analyzed experimentally in white-footed mice and in C3H/HeNCrl mice over a time period of almost 3 months. We found that the initially high transmission efficiency from white-footed mice to ticks declined sharply for isolate B348 but remained considerably high for isolate BL206. In contrast, in C3H/HeNCrl mice, high transmission efficiency persisted for both isolates. Our findings provide proof-of-principle evidence for intrinsic fitness variation of B. burgdorferi strains in vertebrate host species, perhaps indicating the beginnings of adaptive radiation.