Viruses, Volume 13, Issue 4 (April 2021) – 183 articles

In late 2019, the first herpesvirus in the genus Lepus, named leporid gammaherpesvirus 5 (LeHV-5) was described. At the time, herpetic typical lesions were observed in hares infected by the myxoma virus, which is known to induce immunosuppression. Though the real impact of LeHV-5 is still poorly understood, since it affects reproduction, it poses an additional threat to the already fragile populations of Iberian hare, demanding prevalence investigations. In this article, we describe the first quantitative molecular method for LeHV-5 detection, using either Taqman or the EvaGreen systems. This method has excellent sensitivity and specificity, it is able to detect 2.1 copies of LeHV-5 DNA and was validated with an internal control targeting the 18S rRNA gene, allowing monitoring extraction and PCR amplification efficiencies. Full article

Complications of hepatitis C virus (HCV) chronic infection cause ~400,000 deaths worldwide annually. One complication, liver fibrosis, is influenced by host genetic factors. Genes influencing fibrosis include immune, metabolic, oxidative stress, and viral entry genes, such as interleukin 10 (IL10), microsomal triglyceride-transfer protein (MTP), superoxide dismutase-2 (SOD2), and apolipoprotein E (APOE)-encoding genes, respectively. Thus, correlating variations in these genes with HCV-induced fibrosis represents an attractive biomarker for the prognosis of fibrosis severity in chronically infected patients. Here, we aimed to test whether polymorphisms in IL10, MTP, SOD2, and APOE genes correlated with the severity of fibrosis induced by HCV genotype 4 (HCV-gt4) in a cohort of chronically infected Egyptian patients. Our results demonstrate a significant association between the severity of fibrosis and specific SNPs in IL-10, SOD2, and ApoE-encoding genes. Haplotype-combination analysis for IL10, MTP, SOD2, and APOE showed statistically significant associations between specific haplotype combinations and fibrosis severity. Identifying biomarkers correlating with the severity of HCV-gt4-induced fibrosis would significantly impact precision prophylaxis and treatment of patients at risk. Full article

SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced ‘COVID-19 like-illness’, and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen^® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. A pVNT correlated strongly with SARS-CoV-2 VNT and was able to effectively discriminate SARS-CoV-2 antibody positive and negative serum with the same efficiency as the VNT. Moreover, the pVNT was performed with the same level of discrimination across multiple separate institutions. Therefore, the pVNT is a sensitive, specific, and reproducible lower biosafety level alternative to VNT for detecting SARS-CoV-2 antibodies for diagnostic and research applications. Our data illustrate the potential utility of applying VNT or pVNT and ELISA antibody tests in parallel to enhance the sensitivity of exposure to infection. Full article

Dengue virus (DENV) infection, which causes dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, is a severe global health problem in tropical and subtropical areas. There is no effective vaccine or drug against DENV infection. Thus, the development of anti-DENV agents is imperative. This study aimed to assess the anti-DENV activity of (E)-guggulsterone using a DENV infectious system. A specific inhibitor targeting signal molecules was used to evaluate the molecular mechanisms of action. Western blotting and qRT-PCR were used to determine DENV protein expression and RNA replication, respectively. Finally, an ICR suckling mouse model was used to examine the anti-DENV activity of (E)-guggulsterone in vivo. A dose-dependent inhibitory effect of (E)-guggulsterone on DENV protein synthesis and RNA replication without cytotoxicity was observed. The mechanistic studied revealed that (E)-guggulsterone stimulates Nrf2-mediated heme oxygenase-1 (HO-1) expression, which increases the antiviral interferon responses and downstream antiviral gene expression by blocking DENV NS2B/3B protease activity. Moreover, (E)-guggulsterone protected ICR suckling mice from life-threatening DENV infection. These results suggest that (E)-guggulsterone can be a potential supplement for controlling DENV replication. Full article

Confirming ZIKV congenital infection is challenging because viral RNA is infrequently detected. We compared the presence of anti-ZIKV-IgM and the persistence of anti-ZIKV-IgG antibodies over 18 months in two cohorts of infants born to ZIKV-infected mothers: Cohort one: 30 infants with typical microcephaly or major brain abnormalities (Congenital Zika Syndrome-CZS); Cohort two: 123 asymptomatic infants. Serum samples obtained within 6 months of age were tested for anti-ZIKV-IgM. Anti-ZIKV-IgG was quantified in sequential samples collected at birth, 3–6 weeks, 3, 6, 12, and 18 months. ZIKV-RNA was never detected postnatally. Anti-ZIKV-IgM antibodies were detected at least once in 15/25 (60.0%; 95%CI: 38.7–78.9) infants with CZS and in 2/115 (1.7%; 95%CI: 0.2–6.1) asymptomatic infants. Although anti-ZIKV-IgG was always positive within 3–6 weeks of age, IgG levels decreased similarly over time in both cohorts. IgG levels decreased similarly in ZIKV-IgM-positive and ZIKV-IgM-negative CZS infants. Differently from other congenital infections, IgM would fail to diagnose 40% of severely symptomatic infants, and the persistence of IgG is not a useful marker for discriminating congenital infection among infants exposed to maternal ZIKV infection. Full article

The purpose of this paper is to describe the hematological profile of pregnant women with suspected Zika virus (ZIKV) infection followed up at a reference service for infectious diseases in Manaus, Brazil, through a clinical, epidemiological, cross-sectional study of pregnant women with an exanthematic manifestation who looked for care between 2015 and 2017. The participants were 499 pregnant women, classified into four subgroups, according to laboratory confirmation of infections: ZIKV-positive; ZIKV-positive and positive for another infection; positive for another infection but not ZIKV-positive; and not positive for any of the infections investigated. Hematological parameters were analyzed descriptively. The association between maternal infection and the hematological profile, along with the association between the maternal hematological profile and the gestational outcome, were tested. Similar hematic and platelet parameters were observed among pregnant women. However, a significant association was observed between low maternal lymphocyte count and a positive diagnosis for ZIKV (p < 0.001). The increase in maternal platelet count and the occurrence of unfavorable gestational outcome were positively associated. A similar hematic and platelet profile was identified among pregnant women, differing only in the low lymphocyte count among ZIKV-positive pregnant women. Regarding gestational outcomes, in addition to the damage caused by ZIKV infection, altered maternal platelets may lead to unfavorable outcomes, with the need for adequate follow-up during prenatal care. Full article

Rift Valley fever phlebovirus (RVFV) infects humans and a wide range of ungulates and historically has caused devastating epidemics in Africa and the Arabian Peninsula. Lesions of naturally infected cases of Rift Valley fever (RVF) have only been described in detail in sheep with a few reports concerning cattle and humans. The most frequently observed lesion in both ruminants and humans is randomly distributed necrosis, particularly in the liver. Lesions supportive of vascular endothelial injury are also present and include mild hydropericardium, hydrothorax and ascites; marked pulmonary congestion and oedema; lymph node congestion and oedema; and haemorrhages in many tissues. Although a complete understanding of RVF pathogenesis is still lacking, antigen-presenting cells in the skin are likely the early targets of the virus. Following suppression of type I IFN production and necrosis of dermal cells, RVFV spreads systemically, resulting in infection and necrosis of other cells in a variety of organs. Failure of both the innate and adaptive immune responses to control infection is exacerbated by apoptosis of lymphocytes. An excessive pro-inflammatory cytokine and chemokine response leads to microcirculatory dysfunction. Additionally, impairment of the coagulation system results in widespread haemorrhages. Fatal outcomes result from multiorgan failure, oedema in many organs (including the lungs and brain), hypotension, and circulatory shock. Here, we summarize current understanding of RVF cellular tropism as informed by lesions caused by natural infections. We specifically examine how extant knowledge informs current understanding regarding pathogenesis of the haemorrhagic fever form of RVF, identifying opportunities for future research. Full article

Coronavirus-like organisms have been previously identified in Arthropod ectoparasites (such as ticks and unfed cat flea). Yet, the question regarding the possible role of these arthropods as SARS-CoV-2 passive/biological transmission vectors is still poorly explored. In this study, we performed in silico structural and binding energy calculations to assess the risks associated with possible ectoparasite transmission. We found sufficient similarity between ectoparasite ACE and human ACE2 protein sequences to build good quality 3D-models of the SARS-CoV-2 Spike:ACE complex to assess the impacts of ectoparasite mutations on complex stability. For several species (e.g., water flea, deer tick, body louse), our analyses showed no significant destabilisation of the SARS-CoV-2 Spike:ACE complex, suggesting these species would bind the viral Spike protein. Our structural analyses also provide structural rationale for interactions between the viral Spike and the ectoparasite ACE proteins. Although we do not have experimental evidence of infection in these ectoparasites, the predicted stability of the complex suggests this is possible, raising concerns of a possible role in passive transmission of the virus to their human hosts. Full article

Infectious laryngotracheitis (ILT) is an acute, highly contagious infectious disease of the upper respiratory tract in chickens and other poultry species that causes significant economic losses in countries worldwide. Between 2017 and 2019, seven outbreaks of mild to severe respiratory disorders with high suspicion of ILT occurred in commercial and backyard poultry flocks in Slovenia. In all submissions, infection with ILT virus (ILTV) was confirmed by PCR, which is the first report of ILT in Slovenia. Circulating ILT strains were characterized by the sequence and phylogenetic analysis of two fragments of the ICP4 gene. Four strains—three detected in non-vaccinated flocks and one in a flock vaccinated against ILT—were identical or very similar to the chicken embryo–origin live virus vaccines, and the other three were closely related to Russian, Chinese, Australian, and American field strains and to tissue culture origin vaccine strains. As in other diseases, coinfections with other respiratory pathogens in confirmed ILT cases may cause a more severe condition and prolong the course of the disease. In our study, coinfections with Mycoplasma synoviae (7/7 tested flocks), infectious bronchitis virus (5/5 tested flocks), Mycoplasma gallisepticum (4/7 tested flocks), Ornithobacterium rhinotracheale (3/4 tested flocks), and avian pox virus (1/2 tested flocks) were confirmed, indicating the importance of these pathogens in the occurrence of ILT infections. Full article

Various viruses alter nuclear pore complex (NPC) integrity to access the nuclear content favoring their replication. Alteration of the nuclear pore complex has been observed not only in viruses that replicate in the nucleus but also in viruses with a cytoplasmic replicative cycle. In this last case, the alteration of the NPC can reduce the transport of transcription factors involved in the immune response or mRNA maturation, or inhibit the transport of mRNA from the nucleus to the cytoplasm, favoring the translation of viral mRNAs or allowing access to nuclear factors necessary for viral replication. In most cases, the alteration of the NPC is mediated by viral proteins, being the viral proteases, one of the most critical groups of viral proteins that regulate these nucleus–cytoplasmic transport changes. This review focuses on the description and discussion of the role of viral proteases in the modification of nucleus–cytoplasmic transport in viruses with cytoplasmic replicative cycles and its repercussions in viral replication. Full article

Regular tests for human papillomavirus (HPV) and early treatment could represent an important strategy for preventing anal cancers among men who have sex with men (MSM). This study investigated facilitators and barriers to take up clinician-collected and self-collected HPV tests among Chinese MSM. This study was based on the baseline sample of a longitudinal study promoting HPV vaccination among 350 Chinese MSM who had never received an HPV vaccination. The baseline survey was conducted from August 2019 to April 2020. The prevalence of any HPV tests uptake in lifetime was 19.1%; 4.9% had HPV infection in anus, genital, oral cavity, and other places. Among the participants, 20% and 76.8% intended to take up self-financed and free clinician-collected HPV tests, and 76.8% intended to use free self-collected HPV tests. After adjusting for significant background characteristics, perceived risk of HPV infection, and perceived benefits, barriers, cue to action, and self-efficacy related to HPV tests in general and/or specific to self-collected HPV tests were associated with behavioral intention to take up free clinician-collected and/or self-collected HPV tests. Less than 20% of Chinese MSM reported HPV tests uptake. Modifying perceptions related to HPV tests may be useful to increase HPV tests coverage in this group. Full article

Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, Bombyx mori pupae were used as a new platform and infected with recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)–insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 10⁴ vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 10¹² VG/larva and 4.6 × 10¹² VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors. Full article

The nuclear envelope (NE) of eukaryotic cells has a highly structural architecture, comprising double lipid-bilayer membranes, nuclear pore complexes, and an underlying nuclear lamina network. The NE structure is held in place through the membrane-bound LINC (linker of nucleoskeleton and cytoskeleton) complex, spanning the inner and outer nuclear membranes. The NE functions as a barrier between the nucleus and cytoplasm and as a transverse scaffold for various cellular processes. Epstein–Barr virus (EBV) is a human pathogen that infects most of the world’s population and is associated with several well-known malignancies. Within the nucleus, the replicated viral DNA is packaged into capsids, which subsequently egress from the nucleus into the cytoplasm for tegumentation and final envelopment. There is increasing evidence that viral lytic gene expression or replication contributes to the pathogenesis of EBV. Various EBV lytic proteins regulate and modulate the nuclear envelope structure in different ways, especially the viral BGLF4 kinase and the nuclear egress complex BFRF1/BFRF2. From the aspects of nuclear membrane structure, viral components, and fundamental nucleocytoplasmic transport controls, this review summarizes our findings and recently updated information on NE structure modification and NE-related cellular processes mediated by EBV. Full article

Infectious Hematopoietic Necrosis Virus (IHNV) infects juvenile salmonid fish in conservation hatcheries and aquaculture facilities, and in some cases, causes lethal disease. This study assesses intra-specific variation in the IHNV susceptibility of Chinook salmon (Oncorhynchus tshawytscha) in the Columbia River Basin (CRB), in the northwestern United States. The virulence and infectivity of IHNV strains from three divergent virus genogroups are measured in four Chinook salmon populations, including spring-run and fall-run fish from the lower or upper regions of the CRB. Following controlled laboratory exposures, our results show that the positive control L strain had significantly higher virulence, and the UC and MD strains that predominate in the CRB had equivalently low virulence, consistent with field observations. By several experimental measures, there was little variation in host susceptibility to infection or disease. However, a small number of exceptions suggested that the lower CRB spring-run Chinook salmon population may be less susceptible than other populations tested. The UC and MD viruses did not differ in infectivity, indicating that the observed asymmetric field prevalence in which IHNV detected in CRB Chinook salmon is 83% UC and 17% MD is not due to the UC virus being more infectious. Overall, we report little intra-species variation in CRB Chinook salmon susceptibility to UC or MD IHNV infection or disease, and suggest that other factors may instead influence the ecology of IHNV in the CRB. Full article

The COVID-19 pandemic has infected millions worldwide, leaving a global burden for long-term care of COVID-19 survivors. It is thus imperative to study post-COVID (i.e., short-term) and long-COVID (i.e., long-term) effects, specifically as local and systemic pathophysiological outcomes of other coronavirus-related diseases (such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS)) were well-cataloged. We conducted a comprehensive review of adverse post-COVID health outcomes and potential long-COVID effects. We observed that such adverse outcomes were not localized. Rather, they affected different human systems, including: (i) immune system (e.g., Guillain–Barré syndrome, rheumatoid arthritis, pediatric inflammatory multisystem syndromes such as Kawasaki disease), (ii) hematological system (vascular hemostasis, blood coagulation), (iii) pulmonary system (respiratory failure, pulmonary thromboembolism, pulmonary embolism, pneumonia, pulmonary vascular damage, pulmonary fibrosis), (iv) cardiovascular system (myocardial hypertrophy, coronary artery atherosclerosis, focal myocardial fibrosis, acute myocardial infarction, cardiac hypertrophy), (v) gastrointestinal, hepatic, and renal systems (diarrhea, nausea/vomiting, abdominal pain, anorexia, acid reflux, gastrointestinal hemorrhage, lack of appetite/constipation), (vi) skeletomuscular system (immune-mediated skin diseases, psoriasis, lupus), (vii) nervous system (loss of taste/smell/hearing, headaches, spasms, convulsions, confusion, visual impairment, nerve pain, dizziness, impaired consciousness, nausea/vomiting, hemiplegia, ataxia, stroke, cerebral hemorrhage), (viii) mental health (stress, depression and anxiety). We additionally hypothesized mechanisms of action by investigating possible molecular mechanisms associated with these disease outcomes/symptoms. Overall, the COVID-19 pathology is still characterized by cytokine storm that results to endothelial inflammation, microvascular thrombosis, and multiple organ failures. Full article

This study reports on a fatal case of a captive great grey owl infected with the West Nile virus (WNV) in the zoological garden Košice, eastern Slovakia (Central Europe). The tissue samples of the dead owl were used for virus isolation and genetic characterization. The novel isolate is genetically closer to Hungarian, Greek, and Bulgarian strains from the central/southern European clade of lineage 2 than to the strains previously isolated in Slovakia. Interestingly, it carries NS3-249P, a molecular virulence determinant associated with higher neurovirulence, which has not previously been observed in Slovakia. Subsequent serological investigation of the captive owls revealed additional seropositive animals, indicating local WNV transmission. Although no WNV-positive mosquitoes were found, the presence of the WNV principal vector Culex pipiens complex together with the described fatal case and further serological findings indicate an endemic focus of bird-neurovirulent WNV variant in the area. Full article

The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections. Full article

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While detection of SARS-CoV-2 by polymerase chain reaction with reverse transcription (RT-PCR) is currently used to diagnose acute COVID-19 infection, serological assays are needed to study the humoral immune response to SARS-CoV-2. Anti-SARS-CoV-2 immunoglobulin (Ig)G/A/M antibodies against spike (S) protein and its receptor-binding domain (RBD) were characterized in recovered subjects who were RT-PCR-positive (n = 153) and RT-PCR-negative (n = 55) using an enzyme-linked immunosorbent assay (ELISA). These antibodies were also further assessed for their ability to neutralize live SARS-CoV-2 virus. Anti-SARS-CoV-2 antibodies were detected in 90.9% of resolved subjects up to 180 days post-symptom onset. Anti-S protein and anti-RBD IgG titers correlated (r = 0.5157 and r = 0.6010, respectively) with viral neutralization. Of the RT-PCR-positive subjects, 22 (14.3%) did not have anti-SARS-CoV-2 antibodies; and of those, 17 had RT-PCR cycle threshold (Ct) values > 27. These high Ct values raise the possibility that these indeterminate results are from individuals who were not infected or had mild infection that failed to elicit an antibody response. This study highlights the importance of serological surveys to determine population-level immunity based on infection numbers as determined by RT-PCR. Full article

Infections caused by respiratory syncytial virus (RSV) are a major cause of morbidity and mortality in young children worldwide. Understanding seasonal patterns of region-specific RSV activity is important to guide resource allocation for existing and future treatment and prevention strategies. The decades of excellent RSV surveillance data that are available from the developed countries of the world are incredibly instructive in advancing public health initiatives in those regions. With few exceptions, these developed nations are positioned geographically across temperate regions of the world. RSV surveillance across tropical regions of the world has improved in recent years, but remains spotty, and where available, still lacks the necessary longitudinal data to determine the amount of seasonal variation expected over time. However, existing and emerging data collected across tropical regions of the world do indicate that patterns of infection are often quite different from those so well described in temperate areas. Here, we provide a brief summary regarding what is known about general patterns of RSV disease activity across tropical Asia, Africa and South America, then offer additional country-specific details using examples where multiple reports and/or more robust surveillance data have become available. Full article

This paper outlines the role of Lombardy’s regional influenza reference laboratory (Northern Italy) in the surveillance of influenza-like illnesses (ILIs) in monitoring SARS-CoV-2 circulation by analyzing 631 consecutive nasopharyngeal swabs (NPSs) collected from ILI outpatients by sentinel physicians during the 2019–2020 season. The samples were tested by specific real-time RT-PCRs targeting SARS-CoV-2, influenza viruses, and RSVs. Results: Of these NPSs, 31% tested positive for influenza viruses, 10% for SARS-CoV-2, and 7% for RSV. No coinfections were detected. Influenza viruses and RSVs circulated throughout the surveillance period until the end of February (week 9-2020), when they suddenly ceased to circulate seven weeks earlier than during the previous five influenza seasons. After the first detection of SARS-CoV-2 in our ILI outpatients at the beginning of March (week 10-2020), SARS-CoV-2 remained the only virus identified throughout the surveillance period. Patients ≥ 65 years had a 3.2-fold greater risk of being infected with SARS-CoV-2, while school-age children (5–14 years) and children < 5 years proved to be the age groups most at risk of contracting influenza viruses and RSV, respectively. Our experience demonstrates that laboratory-based ILI surveillance networks are essential for identifying SARS-CoV-2 cases that would otherwise remain undetected, in order to stop their spread within our communities. Full article

The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus Curtovirus, which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental Rhizobium-mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated. Full article

Detection of severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) during the early phase of the disease is important for appropriate treatment, infection control, and prevention of further transmission. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification method that amplifies the target sequence under isothermal conditions. Here, we developed an RT-LAMP with a novel primer/probe set targeting a conserved region of the SFTSV L segment after extraction of viral RNA (standard RT-LAMP). Both the Chinese and Japanese SFTSV strains, including various genotypes, were detected by the standard RT-LAMP. We also performed RT-LAMP using the same primer/probe set but without the viral RNA extraction step (called simplified RT-LAMP) and evaluated the diagnostic efficacy. The sensitivity and specificity of the simplified RT-LAMP were 84.9% (45/53) and 89.5% (2/19), respectively. The simplified RT-LAMP can detect SFTSV in human sera containing >10^3.5 copies/mL viral RNA. The two RT-LAMP positive but quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) negative samples were positive in the conventional RT-PCR, suggesting that there was no false positive reaction in the RT-LAMP. Both the standard and simplified RT-LAMP are useful for detecting the SFTSV genome in patients during the early phase of the disease. Full article

Respiratory syncytial virus (RSV) infects most infants by two years of age. It can cause severe disease leading to an increased risk of developing asthma later in life. Previously, our group has shown that RSV infection in mice and infants promotes IL-1β production. Here, we characterized the role of NLRP3-Inflammasome activation during RSV infection in adult mice and neonates. We observed that the inhibition of NLRP3 activation using the small molecule inhibitor, MCC950, or in genetically modified NLRP3 knockout (Nlrp3−/−) mice during in vivo RSV infection led to decreased lung immunopathology along with a reduced expression of the mucus-associated genes and reduced production of innate cytokines (IL-1β, IL-33 and CCL2) linked to severe RSV disease while leading to significant increases in IFN-β. NLRP3-inflammasome inhibition or deletion diminished Th2 cytokines and inflammatory cell infiltration into the lungs. Furthermore, NLRP3 inhibition or deletion during early-life RSV infection led to reducing viral-exacerbated allergic response in a mouse model of RSV-induced allergy exacerbation. Here, we demonstrated the critical role of NLRP3-inflammasome activation in RSV immunopathology and the related long-term airway alteration. Moreover, these findings suggest the NLRP3-inflammasome as a potential therapeutic target to attenuate severe RSV disease and limit childhood asthma development. Full article

Owing to viral evolution and recombination, emerging pseudorabies virus (PRV) strains have caused unprecedented outbreaks in swine farms even when the pigs were previously vaccinated, which might indicate that traditional vaccines were unable to provide effective protection. The development of safe and efficacious vaccines presents prospects to minimize the clinical signs and eventually eradicate the infection. In this study, we used an emerging PRV strain, HNX, as the parental strain to construct a recombinant PRV with TK/gE gene deletion and Fms-related tyrosine kinase 3 ligand (Flt3L) expression, named HNX-TK⁻/gE⁻-Flt3L. HNX-TK⁻/gE⁻-Flt3L enhanced the maturation of bone marrow derived dendritic cells (DCs) in vitro. Significantly more activated DCs were detected in HNX-TK⁻/gE⁻-Flt3L-immunized mice compared with those immunized with HNX-TK⁻/gE⁻. Subsequently, a remarkable increase of neutralizing antibodies, gB-specific IgG antibodies, and interferon-gamma (IFN-γ) was observed in mice vaccinated with HNX-TK⁻/gE⁻-Flt3L. In addition, a lower mortality and less histopathological damage were observed in HNX-TK⁻/gE⁻-Flt3L vaccinated mice with upon PRV lethal challenge infection. Taken together, our results revealed the potential of Flt3L as an ideal adjuvant that can activate DCs and enhance protective immune responses and support the further evaluation of HNX-TK⁻/gE⁻-Flt3L as a promising PRV vaccine candidate. Full article

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57–96.71% protein sequence identity) but shared only 22.65–45.85% and 23.37–43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 10⁴ copies/μL. Full article

Dengue virus (DENV) is a mosquito-borne viral pathogen that plagues many tropical-climate nations around the world, including Brazil. Molecular epidemiology is a growing and increasingly invaluable tool for understanding the dispersal, persistence, and diversity of this impactful virus. In this study, plasma samples (n = 824) from individuals with symptoms consistent with an arboviral febrile illness were analyzed to identity the molecular epidemiological dynamics of DENV circulating in the Brazilian state of Amapá. Twelve DENV type 1 (DENV-1) genomes were identified, which were phylogenetically related to the BR4 lineage of genotype V. Phylodynamics analysis suggested that DENV-1 BR-4 was introduced into Amapá around early 2010, possibly from other states in northern Brazil. We also found unique amino acids substitutions in the DENV-1 envelope and NS5 protein sequences in the Amapá isolates. Characterization of the DENV-1 BR-4 sequences highlights the potential of this new lineage to drive outbreaks of dengue in the Amazon region. Full article

In the plant immune system, according to the ‘gene-for-gene’ model, a resistance (R) gene product in the plant specifically surveils a corresponding effector protein functioning as an avirulence (Avr) gene product. This system differs from other plant–pathogen interaction systems, in which plant R genes recognize a single type of gene or gene family because almost all virus genes with distinct structures and functions can also interact with R genes as Avr determinants. Thus, research conducted on viral Avr-R systems can provide a novel understanding of Avr and R gene product interactions and identify mechanisms that enable rapid co-evolution of plants and phytopathogens. In this review, we intend to provide a brief overview of virus-encoded proteins and their roles in triggering plant resistance, and we also summarize current progress in understanding plant resistance against virus Avr genes. Moreover, we present applications of Avr gene-mediated phenotyping in R gene identification and screening of segregating populations during breeding processes. Full article

Despite great advances in our knowledge of the consequences of Zika virus to human health, many questions remain unanswered, and results are often inconsistent. The small sample size of individual studies has limited inference about the spectrum of congenital Zika manifestations and the prognosis of affected children. The Brazilian Zika Cohorts Consortium addresses these limitations by bringing together and harmonizing epidemiological data from a series of prospective cohort studies of pregnant women with rash and of children with microcephaly and/or other manifestations of congenital Zika. The objective is to estimate the absolute risk of congenital Zika manifestations and to characterize the full spectrum and natural history of the manifestations of congenital Zika in children with and without microcephaly. This protocol describes the assembly of the Consortium and protocol for the Individual Participant Data Meta-analyses (IPD Meta-analyses). The findings will address knowledge gaps and inform public policies related to Zika virus. The large harmonized dataset and joint analyses will facilitate more precise estimates of the absolute risk of congenital Zika manifestations among Zika virus-infected pregnancies and more complete descriptions of its full spectrum, including rare manifestations. It will enable sensitivity analyses using different definitions of exposure and outcomes, and the investigation of the sources of heterogeneity between studies and regions. Full article

Aspergillus and Pseudomonas compete in nature, and are the commonest bacterial and fungal pathogens in some clinical settings, such as the cystic fibrosis lung. Virus infections of fungi occur naturally. Effects on fungal physiology need delineation. A common reference Aspergillus fumigatus strain, long studied in two (of many) laboratories, was found infected with the AfuPmV-1 virus. One isolate was cured of virus, producing a virus-free strain. Virus from the infected strain was purified and used to re-infect three subcultures of the virus-free fungus, producing six fungal strains, otherwise isogenic. They were studied in intermicrobial competition with Pseudomonasaeruginosa. Pseudomonas culture filtrates inhibited forming or preformed Aspergillus biofilm from infected strains to a greater extent, also seen when Pseudomonas volatiles were assayed on Aspergillus. Purified iron-chelating Pseudomonas molecules, known inhibitors of Aspergillus biofilm, reproduced these differences. Iron, a stimulus of Aspergillus, enhanced the virus-free fungus, compared to infected. All infected fungal strains behaved similarly in assays. We show an important consequence of virus infection, a weakening in intermicrobial competition. Viral infection may affect the outcome of bacterial–fungal competition in nature and patients. We suggest that this occurs via alteration in fungal stress responses, the mechanism best delineated here is a result of virus-induced altered Aspergillus iron metabolism. Full article