Intrinsic Immunity Shapes Viral Resistance of Stem Cells.

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.

Covert RNA viruses in medflies differ in their mode of transmission and tissue tropism.

Numerous studies have demonstrated the presence of covert viral infections in insects. These infections can be transmitted in insect populations via two main routes: vertical from parents to offspring, or horizontal between nonrelated individuals. Thirteen covert RNA viruses have been described in the Mediterranean fruit fly (medfly). Some of these viruses are established in different laboratory-reared and wild medfly populations, although variations in the viral repertoire and viral levels have been observed at different time points. To better understand these viral dynamics, we characterized the prevalence and levels of covert RNA viruses in two medfly strains, assessed the route of transmission of these viruses, and explored their distribution in medfly adult tissues. Altogether, our results indicated that the different RNA viruses found in medflies vary in their preferred route of transmission. Two iflaviruses and a narnavirus are predominantly transmitted through vertical transmission via the female, while a nodavirus and a nora virus exhibited a preference for horizontal transmission. Overall, our results give valuable insights into the viral tropism and transmission of RNA viruses in the medfly, contributing to the understanding of viral dynamics in insect populations. IMPORTANCE: The presence of RNA viruses in insects has been extensively covered. However, the study of host-virus interaction has focused on viruses that cause detrimental effects to the host. In this manuscript, we uncovered which tissues are infected with covert RNA viruses in the agricultural pest Ceratitis capitata, and which is the preferred transmission route of these viruses. Our results showed that vertical and horizontal transmission can occur simultaneously, although each virus is transmitted more efficiently following one of these routes. Additionally, our results indicated an association between the tropism of the RNA virus and the preferred route of transmission. Overall, these results set the basis for understanding how viruses are established and maintained in medfly populations.

Identification of Embryonic Chicken Proteases Activating Newcastle Disease Virus and Their Roles in the Pathogenicity of Virus Used as In Ovo Vaccine.

In ovo vaccination is an attractive immunization approach for chickens. However, most live Newcastle disease virus (NDV) vaccine strains used safely after hatching are unsafe as in ovo vaccines due to their high pathogenicity for chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. Our previous studies reported that NDV strain TS09-C was a safe in ovo vaccine, and the F protein cleavage site (FCS) containing three basic amino acids (3B-FCS) was the crucial determinant of the attenuation of TS09-C in chicken embryos. Here, five trypsin-like proteases that activated NDV in chicken embryos were identified. The F protein with 3B-FCS was sensitive to the proteases Tmprss4, Tmprss9, and F7, was present in fewer tissue cells of chicken embryos, which limited the viral tropism, and was responsible for the attenuation of NDV with 3B-FCS, while the F protein with FCS containing two basic amino acids could be cleaved not only by Tmprss4, Tmprss9, and F7 but also by Prss23 and Cfd, was present in most tissue cells, and thereby was responsible for broad tissue tropism and high pathogenicity of virus in chicken embryos. Furthermore, when mixed with the protease inhibitors aprotinin and camostat, NDV with 2B-FCS exhibited greatly weakened pathogenicity in chicken embryos. Thus, our results extend the understanding of the molecular mechanism of NDV pathogenicity in chicken embryos and provide a novel molecular target for the rational design of in ovo vaccines, ensuring uniform and effective vaccine delivery and earlier induction of immune protection by the time of hatching. IMPORTANCE As an attractive immunization approach for chickens, in ovo vaccination can induce a considerable degree of protection by the time of hatching, provide support in closing the window in which birds are susceptible to infection, facilitate fast and uniform vaccine delivery, and reduce labor costs by the use of mechanized injectors. The commercial live Newcastle disease virus (NDV) vaccine strains are not safe for in ovo vaccination and cause the death of chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. In the present study, we identified five trypsin-like proteases that activate NDV in chicken embryos and elucidated their roles in the tissue tropism and pathogenicity of NDV used as in ovo vaccine. Finally, we revealed the molecular basis for the pathogenicity of NDV in chicken embryos and provided a novel strategy for the rational design of in ovo ND vaccines.

Influenza C and D Viruses Demonstrated a Differential Respiratory Tissue Tropism in a Comparative Pathogenesis Study in Guinea Pigs.

Influenza C virus (ICV) is increasingly associated with community-acquired pneumonia (CAP) in children and its disease severity is worse than the influenza B virus, but similar to influenza A virus associated CAP. Despite the ubiquitous infection landscape of ICV in humans, little is known about its replication and pathobiology in animals. The goal of this study was to understand the replication kinetics, tissue tropism, and pathogenesis of human ICV (huICV) in comparison to the swine influenza D virus (swIDV) in guinea pigs. Intranasal inoculation of both viruses did not cause clinical signs, however, the infected animals shed virus in nasal washes. The huICV replicated in the nasal turbinates, soft palate, and trachea but not in the lungs while swIDV replicated in all four tissues. A comparative analysis of tropism and pathogenesis of these two related seven-segmented influenza viruses revealed that swIDV-infected animals exhibited broad tissue tropism with an increased rate of shedding on 3, 5, and 7 dpi and high viral loads in the lungs compared to huICV. Seroconversion occurred late in the huICV group at 14 dpi, while swIDV-infected animals seroconverted at 7 dpi. Guinea pigs infected with huICV exhibited mild to moderate inflammatory changes in the epithelium of the soft palate and trachea, along with mucosal damage and multifocal alveolitis in the lungs. In summary, the replication kinetics and pathobiological characteristics of ICV in guinea pigs agree with the clinical manifestation of ICV infection in humans, and hence guinea pigs could be used to study these distantly related influenza viruses. IMPORTANCE Similar to influenza A and B, ICV infections are seen associated with bacterial and viral co-infections which complicates the assessment of its real clinical significance. Further, the antivirals against influenza A and B viruses are ineffective against ICV which mandates the need to study the pathobiological aspects of this virus. Here we demonstrated that the respiratory tract of guinea pigs possesses specific viral receptors for ICV. We also compared the replication kinetics and pathogenesis of huICV and swIDV, as these viruses share 50% sequence identity. The tissue tropism and pathology associated with huICV in guinea pigs are analogous to the mild respiratory disease caused by ICV in humans, thereby demonstrating the suitability of guinea pigs to study ICV. Our comparative analysis revealed that huICV and swIDV replicated differentially in the guinea pigs suggesting that the type-specific genetic differences can result in the disparity of the viral shedding and tissue tropism.

A 3-month-old neonatal rhesus macaque HFMD model caused by coxsackievirus B1 infection and viral tissue tropism.

Coxsackievirus B1 (CVB1), an enterovirus with multiple clinical presentations, has been associated with potential long-term consequences, including hand, foot, and mouth disease (HFMD), in some patients. However, the related animal models, transmission dynamics, and long-term tissue tropism of CVB1 have not been systematically characterized. In this study, we established a model of CVB1 respiratory infection in rhesus macaques and evaluated the clinical symptoms, viral load, and immune levels during the acute phase (0-14 days) and long-term recovery phase (15-30 days). We also investigated the distribution, viral clearance, and pathology during the long-term recovery period using 35 postmortem rhesus macaque tissue samples collected at 30 days postinfection (d.p.i.). The results showed that the infected rhesus macaques were susceptible to CVB1 and exhibited HFMD symptoms, viral clearance, altered cytokine levels, and the presence of neutralizing antibodies. Autopsy revealed positive viral loads in the heart, spleen, pancreas, soft palate, and olfactory bulb tissues. HE staining demonstrated pathological damage to the liver, spleen, lung, soft palate, and tracheal epithelium. At 30 d.p.i., viral antigens were detected in visceral, immune, respiratory, and muscle tissues but not in intestinal or neural tissues. Brain tissue examination revealed viral meningitis-like changes, and CVB1 antigen expression was detected in occipital, pontine, cerebellar, and spinal cord tissues at 30 d.p.i. This study provides the first insights into CVB1 pathogenesis in a nonhuman primate model of HFMD and confirms that CVB1 exhibits tissue tropism following long-term infection.

The role of PB1-F2 in adaptation of high pathogenicity avian influenza virus H7N7 in chickens.

Avian influenza viruses (AIV) of the H7N7 subtype are enzootic in the wild bird reservoir in Europe, cause infections in poultry, and have sporadically infected humans. The non-structural protein PB1-F2 is encoded in a second open frame in the polymerase segment PB1 and its sequence varies with the host of origin. While mammalian isolates predominantly carry truncated forms, avian isolates typically express full-length PB1-F2. PB1-F2 is a virulence factor of influenza viruses in mammals. It modulates the host immune response, causing immunopathology and increases pro-inflammatory responses. The role of full-length PB1-F2 in IAV pathogenesis as well as its impact on virus adaptation and virulence in poultry remains enigmatic. Here, we characterised recombinant high pathogenicity AIV (HPAIV) H7N7 expressing or lacking PB1-F2 in vitro and in vivo in chickens. In vitro, full-length PB1-F2 modulated viability of infected chicken fibroblasts by limiting apoptosis. In chickens, PB1-F2 promoted gastrointestinal tropism, as demonstrated by enhanced viral replication in the gut and increased cloacal shedding. PB1-F2's effects on cellular immunity however were marginal. Overall, chickens infected with full-length PB1-F2 virus survived for shorter periods, indicating that PB1-F2 is also a virulence factor in bird-adapted viruses.

Coinfection of slime feather duster worms (Annelida, Myxicola) by different gregarine apicomplexans (Selenidium) and astome ciliates reflects spatial niche partitioning and host specificity.

Individual organisms can host multiple species of parasites (or symbionts), and one species of parasite can infect different host species, creating complex interactions among multiple hosts and parasites. When multiple parasite species coexist in a host, they may compete or use strategies, such as spatial niche partitioning, to reduce competition. Here, we present a host­symbiont system with two species of Selenidium (Apicomplexa, Gregarinida) and one species of astome ciliate co-infecting two different species of slime feather duster worms (Annelida, Sabellidae, Myxicola) living in neighbouring habitats. We examined the morphology of the endosymbionts with light and scanning electron microscopy (SEM) and inferred their phylogenetic interrelationships using small subunit (SSU) rDNA sequences. In the host 'Myxicola sp. Quadra', we found two distinct species of Selenidium; S. cf. mesnili exclusively inhabited the foregut, and S. elongatum n. sp. inhabited the mid to hindgut, reflecting spatial niche partitioning. Selenidium elongatum n. sp. was also present in the host M. aesthetica, which harboured the astome ciliate Pennarella elegantia n. gen. et sp. Selenidium cf. mesnili and P. elegantia n. gen. et sp. were absent in the other host species, indicating host specificity. This system offers an intriguing opportunity to explore diverse aspects of host­endosymbiont interactions and competition among endosymbionts.

The Sialyl Lewis X Glycan Receptor Facilitates Infection of Subtype H7 Avian Influenza A Viruses.

Subtype H7 avian influenza A viruses (IAVs) are enzootic in wild aquatic birds and have caused sporadic spillovers into domestic poultry and humans. Here, we determined the distribution of fucosylated α2,3 sialoglycan (i.e., sialyl Lewis X [SLeX]) in chickens and five common dabbling duck species and the association between SLeX and cell/tissue/host tropisms of H7 IAVs. Receptor binding analyses showed that H7 IAVs bind to both α2,3-linked (SA2,3Gal) and α2,6-linked sialic acids (SA2,6Gal), but with a higher preference for SLeX; H7 IAVs replicated more efficiently in SLeX-overexpressed than SLeX-deficient MDCK cells. While chickens and all tested dabbling ducks expressed abundant SA2,3Gal and SA2,6Gal, SLeX was detected in both respiratory and gastrointestinal tissues of chickens and mallard ducks and in only the respiratory tissues of gadwall, green-wing teal, and northern shoveler but not in wood ducks. Viral-tissue binding assays showed that H7 IAVs bind to chicken colon crypt cells that express SLeX but fewer bind to mallard colon crypt cells, which do not express SLeX; H7 IAVs bind efficiently to epithelial cells of all tissues expressing SA2,3Gal. High viral replication was identified in both chickens and mallards infected with an H7 virus, regardless of SLeX expression, and viruses were detected in all cells to the same degree as viruses detected in the viral-tissue binding assays. In summary, this study suggests that SLeX facilitates infection of H7 viruses, but other types of SA2,3Gal glycan receptors shape the tissue/host tropisms of H7 IAVs. IMPORTANCE In addition to causing outbreaks in domestic poultry, subtype H7 IAVs can cause sporadic spillover infections in lower mammals and humans. In this study, we showed that SLeX expression varies among wild dabbling ducks. Although it facilitated virus binding and affected infection of H7 IAV in cells, SLeX expression is not the only determinant of viral replication at either the tissue or host level. This study suggested that access to heterologous SA2,3Gal glycan receptors, including fucosylated α2,3-linked sialoglycans, shape tissue and host tropism of H7 IAVs in aquatic wild birds.

Prevalence of novel gamma HPV types 223 and 225 in oral cavity and skin of Indian normal and neoplastic participants.

Gamma-papillomaviruses, though traditionally classified as cutaneotropic, actual tissue tropism is largely unexplored. This study aimed to evaluate the tissue-specific prevalence of two novel-HPV 223 and 225 in samples of oral mucosa and keratinized epithelium of varied skin parts from 226 female and male subjects, with or without neoplastic/dysplastic lesions in oral cavity or cervix. The gamma-human papillomavirus (gamma-HPV) 223 and 225 DNA presences were determined by polymerase chain reaction (PCR) ursing the HPV type-specific primers and confirmed by Sanger sequencing. Viral load in the HPV 223 and HPV 225 positive samples were determined by absolute real-time quantification method. Alpha-HPV DNA prevalence was also checked in oral mucosa to ascertain coinfection status. Novel HPV 223 was present in 4.4% (10/226) oral mucosal samples of the study population; interestingly all were females with no prevalence in their corresponding skin swab samples. Whereas, the prevalence of HPV 225 was found both in the skin and oral mucosa of 28.2% (N = 37/131) female and 17.9% (N = 17/95) male participants. Alongside, HPV 223 viral load was found to be significantly higher (p = 0.02 < 0.05) in the oral mucosa of diseased participants, whereas, HPV 225 viral load was higher in the oral mucosa of normal participants. Our results suggest that gamma-HPV 223 has its prevalence only in the oral mucosal epithelium, whereas, HPV 225 has its prevalence on both mucosal and keratinized skin epithelium, indicating its dual tropism nature.

A pathogenic and recombinant infectious bronchitis virus variant (CK/CH/GX/202109) with multiorgan tropism.

Despite vaccine use, novel strains and variants of infectious bronchitis virus (IBV) have emerged continuously, leading to economic losses to the poultry industry worldwide. This study aimed to characterize the IBV isolate CK/CH/GX/202109 from three yellow broilers in Guangxi, China. Recombination was shown to have occurred in regions of the 1ab gene. Compared to the whole genome of ck/CH/LGX/130530, which is genotypically related to tl/CH/LDT3-03, the 202109 strain had 21 mutations. The pathological assessment showed that this variant caused 30% and 40% mortality in 1-day-old chicks infected with oral and ocular inoculum, respectively. Nephritis, enlarged proventriculus, inflammation of the gizzard, and atrophy of the bursa of Fabricius were also observed at both 7 and 14 days post-infection (dpi). Viral loads in the trachea, proventriculus, gizzard, kidney, bursa, and cloacal swabs were higher at 7 dpi than at 14 dpi. Clinicopathological and immunohistochemical analyses revealed that this virus exhibited multiple organ tropisms capable of infecting the trachea, proventriculus, gizzard, kidney, bursa, ileum, jejunum, and rectum. Almost none of the 1-day-old infected chicks seroconverted until 14 dpi. While the virus was found in the ileum, jejunum, and rectum in the 28-day-old ocular group, the majority of 28-day-old infected chickens seroconverted at 10 dpi. These study findings demonstrate that recombination events and mutations during the evolution of IBV may greatly alter tissue tropism and emphasize the need for the continued surveillance of novel strains and variants in order to control this infection.

Production and characterization of monoclonal antibodies against mandarinfish ranavirus and first identification of pyloric caecum as the major target tissue.

Mandarinfish ranavirus (MRV), also known as a variant of largemouth bass virus (LMBV), is an emerging pathogen in mandarinfish aquaculture. In this study, monoclonal antibodies (mAbs) against MRV were produced and characterized, and 7 mAbs were obtained through Western blotting screening and all 7 mAbs specifically recognized MRV/LMBV but not several piscine iridoviruses as ISKNV, GIV and TFV. By LC MS/MS analysis, the recognized viral proteins by seven mAbs were identified as MRV-pORF47L, MRV-pORF55R, MRV-pORF57L, MRV-pORF77L and MRV-pORF78L, respectively, and all five viral proteins are late expression structural proteins by Western blotting. Based on mAb 1C4, immuno-histochemistry and immuno-histo-fluorescence were performed to re-assess the tissue tropism of MRV. The result showed that abundant reactive signals were observed in infected spleen, kidney as well as intestine and pyloric caecum. Real-time quantitative PCR also demonstrated that spleen as well as pyloric caecum and intestines are the major target tissue upon MRV infection. In infected intestines and pyloric caecum, numerous enlarged, multinucleated cells with intracytoplasmic inclusions were identified as the target cells of MRV, suggesting that MRV serves as a digestive tract pathogen to mandarinfish, which may explain why acute infection of MRV can cause the typical clinicopathology featured by severe ascites.

Investigation on Pathological Aspects, Mode of Transmission, and Tissue Tropism of Antheraea proylei Nucleopolyhedrovirus Infecting Oak Tasar Silkworm.

The temperate oak tasar silkworm, Antheraea proylei, is frequently infested with Antheraea proylei nucleopolyhedrovirus (AnprNPV) causing tiger band disease. This disease is one of the key factors that obstructs production and productivity of oak tasar sericulture. The current study aimed to investigate the pathogenicity of AnprNPV, its mode of transmission, and detection of AnprNPV in different tissues. Transmission electron micrographs of AnprNPV showed single rod-shaped bodies and occlusion derived virus (ODV) enclosed within multiple envelopes. The infecting AnprNPV displayed tissue tropism with higher copy numbers detected in the insect fat body and ovary. The virus was observed to multiply in all developmental stages of the silkworm such as egg, larva, pupa, and moth, confirming its ability to spread throughout the silkworm lifecycle. Baculovirus isolated from infected A. proylei showed cross-infectivity in other Saturniidae wild silkworm species such as Antheraea pernyi, A. frithi, and Samia ricini, widening their probable host range for infection. Baculoviruses generally display a horizontal mode of transmission, mainly through ingestion of occlusion bodies (OBs); however, the present study revealed a trans-ovum vertical mode of transmission in addition to a horizontal mode. The observations made in this study aid a detailed understanding of the tiger band disease and its causative pathogen AnprNPV, which will support future studies and disease management in oak tasar sericulture.

Genomic Determinants Potentially Associated with Clinical Manifestations of Human-Pathogenic Tick-Borne Flaviviruses.

The tick-borne flavivirus group contains at least five species that are pathogenic to humans, three of which induce encephalitis (tick-borne encephalitis virus, louping-ill virus, Powassan virus) and another two species induce hemorrhagic fever (Omsk hemorrhagic fever virus, Kyasanur Forest disease virus). To date, the molecular mechanisms responsible for these strikingly different clinical forms are not completely understood. Using a bioinformatic approach, we performed the analysis of each amino acid (aa) position in the alignment of 323 polyprotein sequences to calculate the fixation index (Fst) per site and find the regions (determinants) where sequences belonging to two designated groups were most different. Our algorithm revealed 36 potential determinants (Fst ranges from 0.91 to 1.0) located in all viral proteins except a capsid protein. In an envelope (E) protein, most of the determinants were located on the virion surface regions (domains II and III) and one (absolutely specific site 457) was located in the transmembrane region. Another 100% specific determinant site (E63D) with Fst = 1.0 was located in the central hydrophilic domain of the NS2b, which mediates NS3 protease activity. The NS5 protein contains the largest number of determinants (14) and two of them are absolutely specific (T226S, E290D) and are located near the RNA binding site 219 (methyltransferase domain) and the extension structure. We assume that even if not absolutely, highly specific sites, together with absolutely specific ones (Fst = 1.0) can play a supporting role in cell and tissue tropism determination.

Repair of an Attenuated Low-Passage Murine Cytomegalovirus Bacterial Artificial Chromosome Identifies a Novel Spliced Gene Essential for Salivary Gland Tropism.

The cloning of herpesviruses as bacterial artificial chromosomes (BACs) has revolutionized the study of herpesvirus biology, allowing rapid and precise manipulation of viral genomes. Several clinical strains of human cytomegalovirus (HCMV) have been cloned as BACs; however, no low-passage strains of murine CMV (MCMV), which provide a model mimicking these isolates, have been cloned. Here, the low-passage G4 strain of was BAC cloned. G4 carries an m157 gene that does not ligate the natural killer (NK) cell-activating receptor, Ly49H, meaning that unlike laboratory strains of MCMV, this virus replicates well in C57BL/6 mice. This BAC clone exhibited normal replication during acute infection in the spleen and liver but was attenuated for salivary gland tropism. Next-generation sequencing revealed a C-to-A mutation at nucleotide position 188422, located in the 3' untranslated region of sgg1, a spliced gene critical for salivary gland tropism. Repair of this mutation restored tropism for the salivary glands. Transcriptional analysis revealed a novel spliced gene within the sgg1 locus. This small open reading frame (ORF), sgg1.1, starts at the 3' end of the first exon of sgg1 and extends exon 2 of sgg1. This shorter spliced gene is prematurely terminated by the nonsense mutation at nt 188422. Sequence analysis of tissue culture-passaged virus demonstrated that sgg1.1 was stable, although other mutational hot spots were identified. The G4 BAC will allow in vivo studies in a broader range of mice, avoiding the strong NK cell responses seen in B6 mice with other MCMV BAC-derived MCMVs.IMPORTANCE Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have been cloned as bacterial artificial chromosomes (BACs), which permits rapid and precise manipulation. Low-passage strains of MCMV add to the utility of the mouse model of HCMV infection but do not exist as cloned BACs. This study describes the first such low-passage MCMV BAC. This BAC-derived G4 was initially attenuated in vivo, with subsequent full genomic sequencing revealing a novel spliced transcript required for salivary gland tropism. These data suggest that MCMV, like HCMV, undergoes tissue culture adaptation that can limit in vivo growth and supports the use of BAC clones as a way of standardizing viral strains and minimizing interlaboratory strain variation.

Tissue Tropisms of Avian Influenza A Viruses Affect Their Spillovers from Wild Birds to Pigs.

Wild aquatic birds maintain a large, genetically diverse pool of influenza A viruses (IAVs), which can be transmitted to lower mammals and, ultimately, humans. Through phenotypic analyses of viral replication efficiency, only a small set of avian IAVs were found to replicate well in epithelial cells of the swine upper respiratory tract, and these viruses were shown to infect and cause virus shedding in pigs. Such a phenotypic trait of the viral replication efficiency appears to emerge randomly and is distributed among IAVs across multiple avian species and geographic and temporal orders. It is not determined by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. This study demonstrates that phenotypic variants of viral replication efficiency exist among avian IAVs but that only a few of these may result in viral shedding in pigs upon infection, providing opportunities for these viruses to become adapted to pigs, thus posing a higher potential risk for creating novel variants or detrimental reassortants within pig populations.IMPORTANCE Swine serve as a mixing vessel for generating pandemic strains of human influenza virus. All hemagglutinin subtypes of IAVs can infect swine; however, only sporadic cases of infection with avian IAVs are reported in domestic swine. The molecular mechanisms affecting the ability of avian IAVs to infect swine are still not fully understood. From the findings of phenotypic analyses, this study suggests that the tissue tropisms (i.e., in swine upper respiratory tracts) of avian IAVs affect their spillovers from wild birds to pigs. It was found that this phenotype is determined not by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. In addition, our results show that such a phenotypic trait was sporadically and randomly distributed among IAVs across multiple avian species and geographic and temporal orders. This study suggests an efficient way for assessment of the risk posed by avian IAVs, such as in evaluating their potentials to be transmitted from birds to pigs.

Virus distribution and early pathogenesis of highly pathogenic peste-des-petits-ruminants virus in experimentally infected goats.

INTRODUCTION: Despite causing one of the most dreaded diseases of small ruminants, relatively little is known about the pathogenic events, antigen distribution and the cells responsible for the uptake and transmission of peste-des-petits-ruminants virus (PPRV) during primitive stages of infection. OBJECTIVES: We aimed at deciphering the sequential tissue tropism, pathological events and putative role of M2c macrophages during incubatory, prodromal and invasive stages of PPRV infection. METHODOLOGY: A total of 10 goats were sequentially sacrificed at 1, 2, 3, 4, and 5 days post-infection (dpi, n = 2 per time-point) following intranasal inoculation with a highly virulent strain of PPRV (lineage IV PPRV/Izatnagar/94). Histological evaluation to assess PPRV mediated pathologies, RT-qPCR and immunohistochemistry (IHC) to decipher sequential virus distribution, and dual immunolabelling to determine the role of M2c macrophage in early PPRV uptake and transmission was performed. RESULTS: PPRV/Izatnagar/94 caused major pathologies in the lung tissues. Unprecedentedly, PPRV nucleic acid and antigens were detected in various tissues as early as one dpi. RT-qPCR revealed PPRV in the nasal cavity, trachea, bronchi, tongue and lymph nodes draining these tissues from 1 dpi. IHC affirms cells residing in the lamina propria and submucosa of the respiratory tract and tongue and peribronchiolar areas of lungs as the primary target of PPRV. Following initial replication in the respiratory tract, PPRV is transmitted to the regional lymph nodes where primary viral amplification occurs. After viraemia and secondary replication in generalized lymphoid tissues, PPRV infects and replicates in the epithelial cells. Further, we localized CD163+ M2c macrophages in the goat tissues, but dual IHC elucidated that M2c macrophages do not facilitate uptake and transmission of PPRV during the early stages of infection. CONCLUSION: Our study substantiates the disease establishment process and pathogenesis of PPRV/Izatnagar/94 during the incubatory and prodromal stages of infection. Further, we have also observed M2c macrophage distribution in the goat tissues and demonstrated that they do not pick and transmit PPRV.

Kinetic analysis of pathogenicity and tissue tropism of gyrovirus 3 in experimentally infected chickens.

Gyrovirus 3 (GyV3), the third novel emerging species of the genus Gyrovirus of the Anelloviridae family, has been described in multiple hosts. Epidemiologically, there are suggestions that GyV3 is associated with diarrhea/proventriculitis, however, no direct causal evidence exists between GyV3 infection and specific clinical diseases. Herein, we infected special pathogen-free (SPF) chickens with GyV3, and then assessed the pathogenicity and tissue tropism. The results revealed that GyV3 induced persistent infection characterized by diarrhea, aplastic anemia, immunosuppression, and persistent systemic lymphocytic inflammation. Clinically, the infected chickens presented ruffled feathers, diarrhea, anemia, and weight loss. Aplastic anemia was characterized by progressive depletion of hematopoietic cells in the bone marrow, immunosuppression was associated with atrophy of the thymus, spleen, and bursa of Fabricious, progressive lymphocytic inflammations were characterized by proventriculitis, adrenalitis, pancreatitis, hepatitis, nephritis, and bronchitis. Viral loads of GyV3 in tissues exhibited "M", "N", "W" or "V" type dynamic changes. The highest level of viral loads was reported in bone marrow at 7dpi, followed by the adrenal gland at 2 dpi, the sciatic nerve at 7 dpi, and bile at 35 dpi. The bone marrow and kidney demonstrate the strongest immunostaining of GyV3-VP1 antigen and were suggested as the target tissues of GyV3. Collectively, GyV3 is an immunosuppressive pathogenic virus that targets the bone marrow and kidney in chickens. Exploring the pathogenicity and tissue tropism of GyV3 will guide the basic understanding of the biology of GyV3 and its pathogenesis in chickens.

Zebrafish (Danio rerio) larvae as a model for real-time studies of propagating VHS virus infection, tissue tropism and neutrophil activity.

Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense single-stranded RNA virus that infects more than 140 different fish species. In this study, zebrafish larvae were employed as in vivo model organisms to investigate progression of disease, the correlation between propagation of the infection and irreversibility of disease, cell tropism and in situ neutrophil activity towards the VHSV-infected cells. A recombinant VHSV strain, encoding "tomato" fluorescence (rVHSV-Tomato), was used in zebrafish to be able to follow the progress of the infection in the live host in real-time. Two-day-old zebrafish larvae were injected into the yolk sac with the recombinant virus. The virus titre peaked 96 hr post-infection in zebrafish larvae kept at 18°C, and correlated with 33% mortality and high morbidity among the larvae. By utilizing the transgenic zebrafish line Tg(fli1:GFP)y1 with fluorescently tagged endothelial cells, we were able to demonstrate that the virus initially infected endothelial cells lining the blood vessels. By observing the rVHSV-Tomato infection in the neutrophil reporter zebrafish line Tg(MPX:eGFP)i114 , we inferred that only a subpopulation of the neutrophils responded to the virus infection. We conclude that the zebrafish larvae are suitable for real-time studies of VHS virus infections, allowing in vivo dissection of host-virus interactions at the whole organism level.

IAPV-Induced Paralytic Symptoms Associated with Tachypnea via Impaired Tracheal System Function.

Although it had been reported that Israeli acute paralysis virus (IAPV) can cause systemic infection in honey bees, little is known about how it establishes this infection and results in the typical symptoms, paralysis and trembling. Here, we used our previously constructed IAPV infectious clone to investigate viral loads in different tissues of honey bees and further identify the relation between tissue tropism and paralytic symptoms. Our results showed that tracheae showed a greater concentration of viral abundance than other tissues. The abundance of viral protein in the tracheae was positively associated with viral titers, and was further confirmed by immunological and ultrastructural evidence. Furthermore, higher viral loads in tracheae induced remarkable down-regulation of succinate dehydrogenase and cytochrome c oxidase genes, and progressed to causing respiratory failure of honey bees, resulting in the appearance of typical symptoms, paralysis and body trembling. Our results showed that paralysis symptoms or trembling was actually to mitigate tachypnea induced by IAPV infection due to the impairment of honey bee tracheae, and revealed a direct causal link between paralysis symptoms and tissue tropism. These findings provide new insights into the understanding of the underlying mechanism of paralysis symptoms of honey bees after viral infection and have implications for viral disease prevention and specific therapeutics in practice.

Guinea Fowl Coronavirus Diversity Has Phenotypic Consequences for Glycan and Tissue Binding.

Guinea fowl coronavirus (GfCoV) causes fulminating enteritis that can result in a daily death rate of 20% in guinea fowl flocks. Here, we studied GfCoV diversity and evaluated its phenotypic consequences. Over the period of 2014 to 2016, affected guinea fowl flocks were sampled in France, and avian coronavirus presence was confirmed by PCR on intestinal content and immunohistochemistry of intestinal tissue. Sequencing revealed 89% amino acid identity between the viral attachment protein S1 of GfCoV/2014 and that of the previously identified GfCoV/2011. To study the receptor interactions as a determinant for tropism and pathogenicity, recombinant S1 proteins were produced and analyzed by glycan and tissue arrays. Glycan array analysis revealed that, in addition to the previously elucidated biantennary di-N-acetyllactosamine (diLacNAc) receptor, viral attachment S1 proteins from GfCoV/2014 and GfCoV/2011 can bind to glycans capped with alpha-2,6-linked sialic acids. Interestingly, recombinant GfCoV/2014 S1 has an increased affinity for these glycans compared to that of GfCoV/2011 S1, which was in agreement with the increased avidity of GfCoV/2014 S1 for gastrointestinal tract tissues. Enzymatic removal of receptors from tissues before application of spike proteins confirmed the specificity of S1 tissue binding. Overall, we demonstrate that diversity in GfCoV S1 proteins results in differences in glycan and tissue binding properties.IMPORTANCE Avian coronaviruses cause major global problems in the poultry industry. As causative agents of huge economic losses, the detection and understanding of the molecular determinants of viral tropism are of ultimate importance. Here, we set out to study those parameters and obtained in-depth insight into the virus-host interactions of guinea fowl coronavirus (GfCoV). Our data indicate that diversity in GfCoV viral attachment proteins results in differences in degrees of affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for GfCoV tissue tropism and pathogenesis in guinea fowls.