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Enterovirus D68 (EV-D68) is a single-stranded positive-sense RNA virus, and it is one of the family members of Picornaviridae. Except for EV-D68, the entire family Picornaviridae has been illustrated in literature. EV-D68 was first discovered and isolated in California, USA, in 1962. EV-D68 has resulted in respiratory disorders’ outbreaks among children worldwide, and it has been detected in cases of various neurological diseases such as acute flaccid myelitis (AFM). A recent study documented a higher number of EV-D68 cases associated with AFM in Europe in 2016 compared to the 2014 outbreak. EV-D68 is mainly diagnosed by quantitative PCR, and there is an affirmative strategy for EV-D68 detection by using pan-EV PCR on the untranslated region and/or the VP1 or VP2, followed by sequencing of the PCR products. Serological tests are limited due to cross-reactivity of the antigens between the different serotypes. Many antiviral drugs for EV-D68 have been evaluated and showed promising results. In our review, we discuss the current knowledge about EV-D68 and its role in the development of AFM.

An attempt to summarize the data of available information materials on epidemiological aspects, the state and prospects of prevention and treatment of hemorrhagic fevers was. Hemorrhagic fevers of viral nature-zoonotic diseases caused by viruses containing ribonucleic acid are classified into 4 families: Arenaviridae, Bunyaviridae, Filoviridae and Flaviviridae. They are spread all over the world, and their pathogens are easily transmitted from person to person, thereby spreading quickly enough beyond the main focus of biological infection. That is why the causative agents of hemorrhagic fevers are regarded as highly contagious biological agents, and agents bioterrorism. Unfortunately, there are currently no effective means of specific prevention and treatment of these infections, and therapeutic measures are limited to the use of symptomatic means. In this regard, the search for substances with pronounced antiviral activity against pathogens of hemorrhagic fevers that can effectively protect against these infections, as well as prevent their occurrence and spread is one of the priority areas of research in modern Infectology, and with the involvement of modern achievements in the field of molecular Virology and genetic engineering. The data obtained in this regard allow a more in-depth understanding of the pathogenesis of hemorrhagic fevers, the mechanisms of interaction of the pathogen with the host at the cellular level, the mechanisms of intracellular replication of viruses, the formation of the hosts response to viral invasion and clinical manifestations of diseases.

Alphaviruses (genus Alphavirus; family Togaviridae) are a medically relevant family of viruses that include chikungunya virus and Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology. Traditionally, rescuing virus from an infectious cDNA clone requires propagating plasmids in bacteria, which can result in mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative- rolling circle amplification (RCA), an in vitro technology. We demonstrate that the viral yield of transfected RCA product is comparable to midiprepped plasmid, albeit with a slight delay in kinetics. RCA, however, is cheaper and less time-consuming. Further, sequential RCA did not introduce mutations into the viral genome, subverting the need for glycerol stocks and retransformation. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.

Viral infections are posing a great threat to humanity for the last few years. Among these, Chikungunya which is a mosquito-borne viral infection has produced enormous epidemics around the world after been rebounded. Although this infection shows a low mortality rate, patients suffer from fever, arthralgia, and maculopapular rashes, which reduce the quality of life for several weeks to years. The currently available treatments only provide symptomatic relief based on analgesics, antipyretics, and anti-inflammatory drugs which are nonspecific without satisfactory results. Medicinal plants are a widely accepted source of new molecules for the treatment of infectious diseases including viral infections. The scientific reports, primarily focusing on the anti-chikungunya activity of plant extracts, natural origin pure compounds, and their synthetic analog published from 2011 to 2021, were selected from PubMed, Google Scholar, and Scopus by using related keywords like anti-chikungunya plants, natural antivirals for Chikungunya. The present review decodes scientific reports on medicinal plants against chikungunya virus (CHIKV) infection and demystifies the potential phytoconstituents which reveals that the screening of flavonoids containing plants and phytochemicals showing efficacy against other arbovirus infections, may prove as a potential lead for drug development against CHIKV. The present article also outlines pathogenesis, clinical aspects, molecular virology, and diagnostic approaches of CHIKV infection.

A tool to study SARS-CoV-2 Work with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires high-level biocontainment facilities, making it important to develop safer molecular tools that can potentially be used under less stringent conditions. Self-replicating RNAs known as replicons have long been used to study pathogenic RNA viruses; however, developing replicons to study SARS-SoV-2 has been challenging because of its large genome. Ricardo-Lax et al . used a yeast-based system to construct SARS-CoV-2 replicons that cannot assemble infectious virus because they lack the spike protein required for host cell entry. Transfecting cells with a spike-expressing plasmid and separately with the replicon generates replicon delivery particles (RDPs) that are only capable of one cycle of infection. The replicons and the RDPs can be used in different contexts for drug screening, and viral assays. —VV

Viruses modulate mitochondrial processes during infection to increase biosynthetic precursors and energy output, fueling virus replication. In a surprising fashion, although it triggers mitochondrial fragmentation, the prevalent pathogen human cytomegalovirus (HCMV) increases mitochondrial metabolism through a yet-unknown mechanism. Here, we integrate molecular virology, metabolic assays, quantitative proteomics, and superresolution confocal microscopy to define this mechanism. We establish that the previously uncharacterized viral protein pUL13 is required for productive HCMV replication, targets the mitochondria, and functions to increase oxidative phosphorylation during infection. We demonstrate that pUL13 forms temporally tuned interactions with the mitochondrial contact site and cristae organizing system (MICOS) complex, a critical regulator of cristae architecture and electron transport chain (ETC) function. Stimulated emission depletion superresolution microscopy shows that expression of pUL13 alters cristae architecture. Indeed, using live-cell Seahorse assays, we establish that pUL13 alone is sufficient to increase cellular respiration, not requiring the presence of other viral proteins. Our findings address the outstanding question of how HCMV targets mitochondria to increase bioenergetic output and expands the knowledge of the intricate connection between mitochondrial architecture and ETC function.

The two major mechanisms of plant defense against pathogens including viruses are resistance (the host’s ability to limit pathogen multiplication) and tolerance (the host’s ability to reduce the effect of infection on its fitness regardless of the level of pathogen multiplication) [1]. Tolerance is evolutionary more stable defense response and is effected against a larger spectrum of virus isolates compared to resistance; the selection pressure for emergence of virulent strains is also reduced in tolerant cultivars compared to resistant one, and persistent virus infection can improve the plant resilience in sub-optimal environmental conditions [2]. On the basis of our preliminary results [3, 4] we hypothesized that the plant tolerant reaction against virus infection may be in particular the consequence of transcriptomic reprogramming of autophagy and RNA-decay, the key processes of innate plant immunity. So, the task of this study is the experimental verification of this hypothesis by quantitative real-time PCR ascertainment of the autophagy and ROC (RNA quantity control) factors expression changes during persistent shallot virus X infection [5]. The research has been conducted at Molecular Virology Laboratory and Center of collective sharing of the All-Russian Research Institute of Agricultural Biotechnology. In this context the autophagy and RNA-decay factors coding transcripts have been identified and their representation levels were determined in shallot plants. It was drawn a conclusion that persistent Shallot virus infection correlates with the specific autophagy and ROC factors expression changes. Further research on the molecular mechanisms associated with tolerance may identify novel targets for engineering tolerance to improve agriculture practices.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne human pathogen that causes chikungunya fever, which is typically accompanied by severe joint pain. In Asia, serological evidence indicated that CHIKV first emerged in 1954. From the 1950’s to 2005, sporadic CHIKV infections were attributed to the Asian genotype. However, the massive outbreak of CHIKV in India and the Southwest Indian Ocean Islands in 2005 has since raised chikungunya as a worldwide public health concern. The virus is spreading globally, but mostly in tropical and subtropical regions, particularly in South and Southeast Asia. The emergence of the CHIKV East/Central/South African genotype-Indian Ocean lineage (ECSA-IOL) has caused large outbreaks in South and Southeast Asia affected more than a million people over a decade. Notably, the massive CHIKV outbreaks before 2016 and the more recent outbreak in Asia were driven by distinct ECSA lineages. The first significant CHIKV ECSA strains harbored the Aedes albopictus-adaptive mutation E1: A226V. More recently, another mass CHIKV ECSA outbreak in Asia started in India and spread beyond South and Southeast Asia to Kenya and Italy. This virus lacked the E1: A226V mutation but instead harbored two novel mutations (E1: K211E and E2: V264A) in an E1: 226A background, which enhanced its fitness in Aedes aegypti. The emergence of a novel ECSA strain may lead to a more widespread geographical distribution of CHIKV in the future. This review summarizes the current CHIKV situation in Asian countries and provides a general overview of the molecular virology, disease manifestation, diagnosis, prevalence, genotype distribution, evolutionary relationships, and epidemiology of CHIKV infection in Asian countries over the past 65 years. This knowledge is essential in guiding the epidemiological study, control, prevention of future CHIKV outbreaks, and the development of new vaccines and antivirals targeting CHIKV.

The coronavirus disease 2019 (COVID-19) pandemic underscores the need to better understand animal-to-human transmission of coronaviruses and adaptive evolution within new hosts. We scanned more than 182,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes for selective sweep signatures and found a distinct footprint of positive selection located around a non-synonymous change (A1114G; T372A) within the spike protein receptor-binding domain (RBD), predicted to remove glycosylation and increase binding to human ACE2 (hACE2), the cellular receptor. This change is present in all human SARS-CoV-2 sequences but not in closely related viruses from bats and pangolins. As predicted, T372A RBD bound hACE2 with higher affinity in experimental binding assays. We engineered the reversion mutant (A372T) and found that A372 (wild-type [WT]-SARS-CoV-2) enhanced replication in human lung cells relative to its putative ancestral variant (T372), an effect that was 20 times greater than the well-known D614G mutation. Our findings suggest that this mutation likely contributed to SARS-CoV-2 emergence from animal reservoirs or enabled sustained human-to-human transmission., Graphical abstract, A non-synonymous change (T372A) within the spike protein RBD of human SARS-CoV-2 shows higher binding affinity to hACE2 and enhanced replication in human lung cells compared with its putative ancestral variant (T372), providing evidence of a viral mutation that is likely to have been necessary to enable human-to-human transmission.

In less than two decades, three deadly zoonotic coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have emerged in humans, causing SARS, MERS, and coronavirus disease 2019 (COVID-19), respectively. The current COVID-19 pandemic poses an unprecedented crisis in health care and social and economic development. It reinforces the cruel fact that CoVs are constantly evolving, possessing the genetic malleability to become highly pathogenic in humans. In this review, we start with an overview of CoV diseases and the molecular virology of CoVs, focusing on similarities and differences between SARS-CoV-2 and its highly pathogenic as well as low-pathogenic counterparts. We then discuss mechanisms underlying pathogenesis and virus-host interactions of SARS-CoV-2 and other CoVs, emphasizing the host immune response. Finally, we summarize strategies adopted for the prevention and treatment of CoV diseases and discuss approaches to develop effective antivirals and vaccines.

Seven human coronaviruses (HCoVs) have been so far identified, namely HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and the novel coronavirus (2019-nCoV, a.k.a. SARS-CoV-2). Unlike the highly pathogenic SARS-CoV, MERS-CoV, and 2019-nCoV, the four so-called common HCoVs generally cause mild upper-respiratory tract illness and contribute to 15%–30% of cases of common colds in human adults, although severe and life-threatening lower respiratory tract infections can sometimes occur in infants, elderly people, or immunocompromised patients. In this article, we review the molecular virology of these common HCoVs, and summarize current knowledge on HCoV-host interaction, pathogenesis, and other clinically relevant perspectives.

Hepatitis B virus (HBV) is the prototypic member of Orthohepadnaviridae, hepadnaviruses that can lead to transient or persistent infection. When left untreated, chronic HBV infection leads to severe liver damage culminating in hepatocellular carcinoma (HCC). HCC represents the third cause of cancer-related death worldwide with more than 800,000 deaths every year, thus constituting a major health issue.

Brazil has the highest SARS-CoV-2 case-fatality rate in pregnant women in the Americas. In this study, clinical and virological findings of five mildly symptomatic pregnant women and their infected fetuses/newborns treated at a referral hospital for COVID19-pregnant women in Midwestern Brazil are reported. Mother and fetal samples were tested by RT-qPCR, ECLIA and Illumina MiSeq sequencing. From the five cases, one resulted in spontaneous abortion, one was stillborn, two were preterm births and one full-term birth. Maternal and fetal placenta, newborn and stillborn secretions were SARS-CoV-2+; one neonate developed ground-glass opacities in his lungs. One neonate’s umbilical cord was IgG+ and all were IgM negative upon hospital discharge. Genomes recovered from two placentas belong to the B.1.1.28 and B.1.1.33 lineages and present nonsynonymous mutations associated with virus fitness and infectivity; other not frequently reported mutations (B.1.1.33: NSP3 V2090G, M A2S and ORF3ab S253P and Y264N; B.1.1.28: NSP3 E995D, NSP12 R240K, NSP14 H1897Y and in ORF7b V21F) were found in proteins involved in viral replication, viral induction of apoptosis, viral interference on interferon and on NF-Κβ pathways. Phylogeny indicates the south of Brazil as the possible origin of these lineages circulating in MT. These findings contribute to describe SARS-CoV-2 infection and outcomes in pregnant women and their fetuses, at any stage of gestation and even in mild symptomatic cases., Graphical abstract SARS-CoV-2 lineages B.1.1.28 and B.1.1.33 were detected in placenta from two out of five mild symptomatic SARS-CoV-2-infected pregnant women and their fetus/newborns from Midwestern Brazil.Image 1

A novel coronavirus (2019-nCov) emerged in China, at the end of December 2019 which posed an International Public Health Emergency, and later declared as a global pandemic by the World Health Organization (WHO). The International Committee on Taxonomy of Viruses (ICTV) named it SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2), while the disease was named COVID-19 (Coronavirus Disease- 2019). Many questions related to the exact mode of transmission, animal origins, and antiviral therapeutics are not clear yet. Nevertheless, it is required to urgently launch a new protocol to evaluate the side effects of unapproved vaccines and antiviral therapeutics to accelerate the clinical application of new drugs. In this review, we highlight the most salient characteristics and recent findings of COVID-19 disease, molecular virology, interspecies mechanisms, and health consequences related to this disease.

Alphaviruses (genusAlphavirus; familyTogaviridae) are a medically relevant family of viruses that include chikungunya virus, Eastern equine encephalitis virus, and the emerging Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology and to create effective vaccines. The traditional approach to rescuing virus from an infectious cDNA clone requires propagating large amounts of plasmids in bacteria, which can result in unwanted mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative to the bacterial-based plasmid platform that uses rolling circle amplification (RCA), anin vitrotechnology that amplifies plasmid DNA using only basic equipment. We demonstrate that the use of RCA to amplify plasmid DNA is comparable to the use of a midiprepped plasmid in terms of viral yield, albeit with a slight delay in virus recovery kinetics. RCA, however, has lower cost and time requirements and amplifies DNA with high fidelity and with no chance of unwanted mutations due to toxicity. We show that sequential RCA reactions do not introduce mutations into the viral genome and, thus, can replace the need for glycerol stocks or bacteria entirely. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.ImportanceThe development of infectious cDNA clones is critical to studying viral pathogenesis and for developing vaccines. The current method for propagating clones in bacteria is limited by the toxicity of the viral genome within the bacterial host, resulting in deleterious mutations in the viral genome, which can only be detected through whole-genome sequencing. These mutations can attenuate the virus, leading to lost time and resources and potentially confounding results. We have developed an alternative method of preparing large quantities of DNA that can be directly transfected to recover infectious virus without the need for bacteria by amplifying the infectious cDNA clone plasmid using rolling circle amplification (RCA). Our results indicate that viral rescue from an RCA product produces a viral yield equal to bacterial-derived plasmid DNA, albeit with a slight delay in replication kinetics. The RCA platform, however, is significantly more cost and time-efficient compared to traditional approaches. When the simplicity and costs of RCA are combined, we propose that a shift to an RCA platform will benefit the field of molecular virology and could have significant advantages for recombinant vaccine production.

Dengue virus (DENV) and Zika virus (ZIKV) are two mosquito-borne flaviviruses afflicting nearly half of the world population. Human infection by these viruses can either be asymptomatic or manifest as clinical diseases from mild to severe. Despite more cases are presented as self-limiting febrile illness, severe dengue disease can be manifested as hemorrhagic fever and hemorrhagic shock syndrome, and ZIKV infection has been linked to increased incidence of peripheral neuropathy Guillain-Barre syndrome and central neural disease such as microcephaly. The current prevention and treatment of these infectious diseases are either non-satisfactory or entirely lacking. Because DENV and ZIKV have much similarities in genomic and structural features, almost identical mode of mosquito-mediated transmission, and probably the same pattern of host innate and adaptive immunity toward them, it is reasonable and often desirable to investigate these two viruses side-by-side, and thereby devise common countermeasures against both. Here, we review the existing knowledge on DENV and ZIKV regarding epidemiology, molecular virology, protective immunity and vaccine development, discuss recent new discoveries on the functions of flavivirus NS1 protein in viral pathogenesis and transmission, and propose a one-two punch strategy using vaccine and vector blockade to overcome antibody-dependent enhancement and defeat Dengue and Zika viruses.

Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways, including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate host’s IFN-I response, West Nile virus (WNV) replication is sensitive to pretreatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNV’s sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells, WNV replication is significantly increased and IFN-I induction and signaling are impaired compared to wild-type (wt) cells. IFN-β pretreatment was more effective in protecting against subsequent WNV infection in wt cells than TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next-generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced JAK1 and STAT1 phosphorylation and impaired induction of several interferon-stimulated genes (ISGs) following WNV infection or IFN-β treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function are dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identify VAMP8 as a novel regulator of the IFN-I system. IMPORTANCE WNV is a mosquito-borne flavivirus that poses a threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here, we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and virus-targeted antiviral strategies.

Over the past two decades next-generation sequencing (NGS) technologies have revolutionized the world of biological sciences. With every passing day, the decline in the cost of sequencing and the availability of free online software and tools for data analysis has made it possible to quickly sequence and analyze the massive amount of genomic data being generated. NGS has the potential to not only greatly improve the research productivity in a short period of time but can also provide more reliable and effective information for practical purposes. Viruses are small infectious particles that affect not only humans but also plants, thereby causing huge economic losses every year globally. Plant viruses are enormous in number and their mutation rate is extremely high, which makes it nearly impossible to control them. In order to design effective virus-control strategies it is of utmost importance for us to understand viral genomics. NGS technologies offer the most suitable platform for rapid sequencing of plant viruses, making it convenient for the scientific community to have a better understanding of virus replication and pathogenicity. On the other hand primary biological databases, such as GenBank, DNA Data Bank of Japan (DDBJ), and European Molecular Biology Laboratory (EMBL) are playing a crucial role in data retrieval and analysis.

International audience; Pest control in agriculture targeting insects, bacteria, fungi, and viruses appeared to be substantial for reducing economic disaster. While chemists held a place in the control of insects, pathologists shared their experience within the diversity of microbes. Following the division into annual and perennial crops, breeders and pathologists were under pressure to improve crops. Annual crops required more assistance of chemists, so we chose in this chapter to focus on perennials and innovative technologies based onto either genetics or genetic engineering. Long-term research on molecular virology and viral resistance has turned from the classical breeding techniques to genetic engineering. This chapter covers a combination of diversified technologies (classical breeding, virus detection and disease identification, molecular research, including natural gene identification and selection, and genetic engineering of virus resistance) that have emerged from pest control; their efficiency in perennials is discussed here.

Summary There is increasing demand for access to rapid microbiological testing, with a view to improving clinical outcomes. The possibility of rapid testing has been facilitated by development of cartridge-based random access molecular technologies that are now widely available. Whether the expense of cartridge-based assays is justified in terms of clinical or laboratory cost savings is controversial. This prospective study evaluated the impact of the Biofire FilmArray Respiratory Panel (‘FilmArray’), a cartridge-based random access molecular test, compared with standard batched molecular testing using an ‘in-house’ respiratory polymerase chain reaction (PCR) on laboratory and health service outcomes for adult patients at a tertiary-level adult hospital in Melbourne, Australia. Laboratory result turnaround time was significantly reduced with the FilmArray (median 4.4 h) compared to a standard validated in-house respiratory PCR assay (median 21.6 h, p < 0.0001) and there was a significant increase in diagnostic yield with the Filmarray (71/124, 57.3%) compared to in-house PCR (79/200; 39.5%; p = 0.002). Despite improved result turnaround time and increased diagnostic yield from testing, there was no corresponding reduction in hospital length of stay or use of isolation beds. Although cartridge-based molecular testing reduced turnaround time to result for respiratory pathogen testing, it did not impact on health service outcomes such as hospital length of stay. Further work is warranted to determine whether cartridge-based tests at the point of care can improve clinical and health service impacts.

Background: Many details of HIV-1 molecular virology have been translated into lifesaving antiviral drugs. Yet, we have an incomplete understanding of the cells in which HIV-1 replicates in untreated individuals and persists in during antiretroviral therapy.Methods: In this review we discuss how viral entry phenotypes have been characterized and the insights they have revealed about the target cells supporting HIV-1 replication. In addition, we will examine whether some HIV-1 variants have the ability to enter cells lacking CD4 (such as astrocytes) and the role that trans-infection plays in HIV-1 replication.Results: HIV-1 entry into a target cell is determined by whether the viral receptor (CD4) and the coreceptor (CCR5 or CXCR4) are expressed on that cell. Sustained HIV-1 replication in a cell type can produce viral lineages that are tuned to the CD4 density and coreceptor expressed on those cells; a fact that allows us to use Env protein entry phenotypes to infer information about the cells in which a viral lineage has been replicating and adapting.Conclusion: We now recognize that HIV-1 variants can be divided into three classes representing the primary target cells of HIV-1; R5 T cell-tropic variants that are adapted to entering memory CD4+ T cells, X4 T cell-tropic variants that are adapted to entering naïve CD4+ T cells and Mtropic variants that are adapted to entering macrophages and possibly other cells that express low levels of CD4. While much progress has been made, the relative contribution that infection of different cell subsets makes to viral pathogenesis and persistence is still being unraveled.

BACKGROUND HBV DNA and quantitative (q)HBsAg levels as prognostic markers for HBV-related disease are mostly validated in Asia and their significance in Western populations is uncertain. AIM To analyse the impact of the HBV genotype and frequent mutations in precore (PC), basal core promoter (BCP) and preS on HBV DNA and qHBsAg levels. METHODS HBV DNA and qHBsAg serum levels of 465 patients with HBeAg-negative chronic HBV infection were correlated with the HBV genotype and mutations in PC, BCP and preS. For a detailed analysis of the molecular virology, genotype A2 genomes harbouring these mutations were analysed for replication efficacy and HBsAg release in cell culture. RESULTS While no impact of the HBV genotype on HBV DNA levels was observed, qHBsAg levels differed up to 1.4 log among the genotypes (P

Although epidemic jaundice was well known to physicians of antiquity, it is only in recent years that medical science has begun to unravel the origins of hepatitis A virus (HAV) and the unique pathobiology underlying acute hepatitis A in humans. Improvements in sanitation and the successful development of highly efficacious vaccines have markedly reduced the worldwide occurence of this enterically-transmitted infection over the past quarter century, yet the virus persists in vulnerable populations and those without HAV immunity and remains a common cause of food-borne disease outbreaks in economically-advantaged societies. Reductions in HAV incidence have led to increases in the median age at which infection occurs, often resulting in more severe disease in affected persons and paradoxical increases in disease burden in some developing nations. Here, we summarize recent advances in the molecular virology and epidemiology of HAV, an atypical member of the Picornaviridae family, survey what is known of the pathogenesis of hepatitis A in humans and the host-pathogen interactions that typify the infection. The article also reviews medical and public health aspects of HAV vaccination and disease prevention. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

On February 18, 2020, Dr. Gunther Keil passed away unexpectedly, shortly after his retirement. He had worked as a scientist at the Friedrich-Loeffler-Institut (FLI; previously Federal Research Centre for Virus Diseases of Animals) since 1981, first in Tubingen, and since 1995 as deputy director of the Institute of Molecular Virology and Cell Biology on the Isle of Riems.

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple cancers in immunocompromised patients including two lymphoproliferative disorders associated with KSHV infection of B lymphocytes. Despite many years of research into the pathogenesis of KSHV associated diseases, basic questions related to KSHV molecular virology remain unresolved. One such unresolved question is the cellular receptors and viral glycoproteins needed for KSHV entry into primary B lymphocytes. In this study, we assess the contributions of KSHV glycoprotein H (gH) and the cellular receptor DC-SIGN to KSHV infection in tonsil-derived B lymphocytes. Our results show that (1) neither KSHV-gH nor DC-SIGN are essential for entry into any B cell subset, (2) DC-SIGN does play a role in KSHV entry into tonsil-derived B cells, but in all B cell subtypes alternative entry mechanisms exist, (3) KSHV-gH can participate in KSHV entry into centrocytes via a DC-SIGN independent entry mechanism, and (4) in the absence of KSHV-gH, DC-SIGN is required for KSHV entry into centrocytes. Our results provide a first glimpse into the complexity of KSHV entry in the lymphocyte compartment and highlight that multiple subset-dependent entry mechanisms are employed by KSHV which depend upon multiple cellular receptors and multiple KSHV glycoproteins.

Hepatitis C virus (HCV) is one of the most prevalent causes of chronic blood-borne infections worldwide. Despite developments of highly effective treatments, most infected individuals are unaware of their infection. Approximately 75% of infections are in low- and middle-income countries; therefore, continuing research in HCV molecular virology and the development of vaccines and affordable diagnostics is required to reduce the global burden. Various intracellular forms of the HCV nucleocapsid (core) protein are produced in cell culture; these comprise the conventional p21 core and the newly discovered shorter isoforms (minicores). Minicores lack the N-terminus of p21 core. This study was conducted to determine if minicores are secreted in cell culture and more importantly if they circulate in the blood of individuals infected with HCV. We also developed a new monoclonal antibody that detects minicores targeting a C-terminal region common to p21 core and minicores. Direct evidence of minicores requires western blot analysis to distinguish the detection of p21 core from minicores. However, the sensitivity for western blot detection of HCV proteins from blood is nil without their prior purification/enrichment from blood. Therefore, we developed a purification method based on a heparin/Mn+2 precipitation of apolipoprotein B-containing lipoproteins because HCV is thought to circulate as a hybrid lipoviral particle. Minicores are secreted in culture when cells are grown in the presence of human serum. The heparin/Mn+2 precipitate from HCV-infected cell culture supernatants and from the blood of 4 patients with high-titer genotype-1 HCV contained minicores. Conclusion: Minicores are major newly discovered HCV proteins that are secreted and circulate in blood during natural infections. Minicore proteins have translational potential as targets in diagnostic assays and in vaccine development. (Hepatology Communications 2018;2:21-28).

Hepatitis B virus is a member of the Hepadnaviridae family and responsible for causing acute and chronic hepatitis in humans. The current estimates of people chronically infected with the virus are put at 250 million worldwide. Immune-mediated liver damage in these individuals may lead to the development of cirrhosis and hepatocellular carcinoma later in life. This review deals with our current understanding of the virology, molecular biology, life cycle and cell-to-cell spread of this very important pathogen, all of which are considered essential for current and future approaches to antiviral treatment.

Study of the possibilities of virions and viral proteins modifications and structural remodeling is an important problem of the modern molecular virology. A technique of heat treatment of rod-shaped tobacco mosaic virus that allowed producing structurally modified spherical particles consisting of the virus coat protein was previously developed in our laboratory. These particles possessed unique adsorption and immunogenic properties and were successfully used to develop a new candidate vaccine against rubella virus. Later, the possibility of thermal remodeling of the filamentous virions of potato virus X was demonstrated. The present work reports a comparative study of thermal remodeling of viruses with different structure belonging to various taxonomic groups. The generation of structurally modified spherical particles by the heat treatment of rod-shaped virions with helical symmetry (dolichos enation mosaic virus and barley stripe mosaic virus) has been demonstrated. The dependence of the size of spherical particles derived from dolichos enation mosaic virus on the initial virus concentration was revealed. The process of thermal remodeling of the filamentous virions and virus-like particles of alternanthera mosaic virus was studied. Heat treatment of plant viruses with icosahedral symmetry was shown to cause no morphological changes.

In humans, hepatitis D virus (HDV) infection only occurs in the presence of a concomitant hepatitis B virus (HBV) infection, and induces the most severe form of human viral hepatitis. Even though HDV is spread worldwide and is endemic in some regions, screening and treatment has been often neglected in part due to the lack of an effective therapy. Moreover, HDV prevalence rates are increasing in many countries driven by immigration from areas of high endemicity. Currently, no FDA-approved anti-HDV therapy is available, although interferon (IFN) alpha therapy has demonstrated benefit in a minority of patients. In this review, we present a current view of our understanding of the epidemiology, molecular virology and management of HDV infection. We additionally discuss new treatment approaches in development and describe the most promising results of recent and ongoing clinical trials of these new potential agents.

The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral functions work in part via viral transcriptional suppression and that HSV-1 has acquired mechanisms to block its functions via proteasome-dependent degradation.

In the past 10years, there has been a lot of enthusiasm surrounding the use of serum hepatitis B surface antigen (HBsAg) quantification to predict disease activity and monitor treatment response in chronic hepatitis B. The measurement of HBsAg levels have been standardized in IU/ml, and nowadays it is almost a mandatory measurement due to the development of new antiviral treatments aiming at HBsAg seroclearance, i.e., functional cure of hepatitis B. Recently, there has been an improved understanding of the molecular virology of HBsAg, and particularly the relative roles of covalently closed circular DNA and integrated hepatitis B virus (HBV) DNA. This has shed new light on the interpretation of HBsAg levels in different phases of chronic hepatitis B. HBsAg level can assist the differentiation of immune tolerance and immune clearance in hepatitis B e antigen (HBeAg)-positive patients, and it can predict inactive disease and spontaneous HBsAg seroclearance in HBeAg-negative patients. The determination of HBsAg level is pivotal to individualize pegylated interferon (PegIFN) treatment; it is the key investigation to decide early termination of PegIFN among non-responders. Among patients treated by nucleos(t)ide analogues, responders tend to have dramatic reduction of HBsAg to low levels, which may be followed by HBsAg seroclearance. With newer data on combination treatment of PegIFN and nucleos(t)ide analogues as well as emerging new antiviral agents, HBsAg quantification is expected to become increasingly important to monitor and guide antiviral therapy for chronic hepatitis B.

Animal hepaciviruses represent promising surrogate models for hepatitis C virus (HCV), for which there are no efficient immunocompetent animal models. Experimental infection of laboratory rats with rodent hepacivirus isolated from feral Rattus norvegicus (RHV-rn1) mirrors key aspects of HCV infection in humans, including chronicity, hepatitis, and steatosis. Moreover, RHV has been adapted to infect immunocompetent laboratory mice. RHV in vitro systems have not been developed but would enable detailed studies of the virus life cycle crucial for designing animal experiments to model HCV infection. Here, we established efficient RHV-rn1 selectable subgenomic replicons with and without reporter genes. Rat and mouse liver-derived cells did not readily support the complete RHV life cycle, but replicon-containing cell clones could be selected with and without acquired mutations. Replication was significantly enhanced by mutations in NS4B and NS5A and in cell clones cured of replicon RNA. These mutations increased RHV replication of both mono- and bicistronic constructs, and CpG/UpA-dinucleotide optimization of reporter genes allowed replication. Using the replicon system, we show that the RHV-rn1 NS3-4A protease cleaves a human mitochondrial antiviral signaling protein reporter, providing a sensitive readout for virus replication. RHV-rn1 replication was inhibited by the HCV polymerase inhibitor sofosbuvir and high concentrations of HCV NS5A antivirals but not by NS3 protease inhibitors. The microRNA-122 antagonist miravirsen inhibited RHV-rn1 replication, demonstrating the importance of this HCV host factor for RHV. These novel RHV in vitro systems will be useful for studies of tropism, molecular virology, and characterization of virus-host interactions, thereby providing important complements to in vivo systems. IMPORTANCE A vaccine against hepatitis C virus (HCV) is crucial for global control of this important pathogen, which induces fatal human liver diseases. Vaccine development has been hampered by the lack of immunocompetent animal models. Discovery of rodent hepacivirus (RHV) enabled establishment of novel surrogate animal models. These allow robust infection and reverse genetic and immunization studies of laboratory animals, which develop HCV-like chronicity. Currently, there are no RHV in vitro systems available to study tropism and molecular virology. Here, we established the first culture systems for RHV, recapitulating the intracellular phase of the virus life cycle in vitro. These replicon systems enabled identification of replication-enhancing mutations and selection of cells highly permissive to RHV replication, which allow study of virus-host interactions. HCV antivirals targeting NS5A, NS5B, and microRNA-122 efficiently inhibited RHV replication. Hence, several important aspects of HCV replication are shared by the rodent virus system, reinforcing its utility as an HCV model.

Immunoelectron microscopy is a powerful technique for identifying viral antigens and determining their structural localization and organization within vaccines and viruses. While traditional negative staining transmission electron microscopy provides structural information, identity of components within a sample may be confounding. Immunoelectron microscopy allows for identification and visualization of antigens and their relative positions within a particulate sample. This allows for simple qualitative analysis of samples including whole virus, viral components, and viral-like particles. This article describes methods for immunogold labeling of viral antigens in a liquid suspension, with examples of immunogold-labeled influenza virus glycoproteins, and also discusses the important considerations for sample preparation and determination of morphologies. Together, these methods allow for understanding the antigenic makeup of viral particulate samples, which have important implications for molecular virology and vaccine development. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

The triennial International Double-Stranded RNA Virus Symposium, this year organized by J. Matthijnssens, J. S. L. Parker, P. Danthi, and P. Van Damme in Belgium, gathered over 200 scientists to discuss novel observations and hypotheses in the field. The keynote lecture on functional interactions of bacteria and viruses in the gut microbiome was presented by Julie Pfeiffer. Workshops were held on viral diversity, molecular epidemiology, molecular virology, immunity and pathogenesis, virus structure, the viral use and abuse of cellular pathways, and applied double-stranded RNA (dsRNA) virology. The establishment of a plasmid only-based reverse genetics system for rotaviruses by several Japanese research groups in 2017 has now been reproduced by various other research groups and was discussed in detail. The visualization of dsRNA virus replication steps in living cells received much attention. Mechanisms of the cellular innate immune response to virus infection and of viral pathogenesis were explored. Knowledge of the gut microbiome’s influence on specific immune responses has increased rapidly, also due to the availability of relevant animal models of virus infection. The method of cryo-electron microscopic (cryo-EM) tomography has elucidated various asymmetric structures in viral particles. The use of orthoreoviruses for oncolytic virotherapy was critically assessed. The application of llama-derived single chain nanobodies for passive immunotherapy was considered attractive. In a satellite symposium the introduction, impact and further developments of rotavirus vaccines were reviewed. The Jean Cohen Lecturer of this meeting was Harry B. Greenberg, who presented aspects of his research on rotaviruses over a period of more than 40 years. He was also interviewed at the meeting by Vincent Racaniello for the 513th session of This Week in Virology.

Hepatitis C virus (HCV) infection was once considered a threat to life but is now curable. This miraculous achievement is the result of years of effort to understand basic HCV biology, which led to the development of HCV cell culture systems eventually enabling drug discovery. Initial studies focused on biochemical characterization of viral proteins and dissected their roles in the virus life cycle. Two of the viral proteins, NS3-4A protease and NS5B polymerase, were selected early on as potential drug targets, and subsequent collaborative efforts of academia and industry led to the development of highly effective inhibitors against these enzymes. Another HCV protein, NS5A that has no known enzymatic activity, was more recently identified as an unexpected target of a highly potent class of anti-HCV inhibitors. Various combinations of these protease, polymerase, and NS5A inhibitors now constitute the current anti-HCV regimens with cure rates of above 95%. This chapter is divided into two parts. The first part begins with a short introduction to HCV and its life cycle and reviews insights into biochemical and functional characteristics of HCV RNA elements and proteins. The second part discusses the HCV animal models and how their use yielded important insights into the viral life cycle, immunity, and disease pathogenesis.

Plant viruses always posed extensive losses to crop production. Thus, it is of utmost importance to plant virologists and biologists to accurately identify culprit host plant proteins which participate in plant-virus interactions. Advancements in molecular virology and plant biotechnology have led to many major breakthroughs in past years enabling recognition of innumerable host factors of virus-plant interactions. Interestingly majority of these host factors are chloroplast and photosynthesis related proteins. Hence chloroplast-virus interaction is an epicentre of plant-virus interplays, and its study could help to understand mechanisms of virus infection, spread, symptom development and host resistance. Advanced proteomic tools have empowered the development of sensitive and effective methods to detect host and viral proteins of interplays. Thus, precise information on chloroplast-virus interaction could be used to develop finer disease control strategies and genetically engineered plants with better photosynthetic efficiency and yields.

Several recently developed high-throughput techniques have changed the field of molecular virology. For example, proteomics studies reveal complete interactomes of a viral protein, genome-wide CRISPR knockout and activation screens probe the importance of every single human gene in aiding or fighting a virus, and ChIP-seq experiments reveal genome-wide epigenetic changes in response to infection. Deep mutational scanning is a relatively novel form of protein science which allows the in-depth functional analysis of every nucleotide within a viral gene or genome, revealing regions of importance, flexibility, and mutational potential. In this review, we discuss the application of this technique to RNA viruses including members of the Flaviviridae family, Influenza A Virus and Severe Acute Respiratory Syndrome Coronavirus 2. We also briefly discuss the reverse genetics systems which allow for analysis of viral replication cycles, next-generation sequencing technologies and the bioinformatics tools that facilitate this research.

Human papillomavirus (HPV) was proven to play a significant role in cancer development in the oropharynx. However, its role in the development of laryngeal (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) remains to be clarified. High-risk HPV (HR-HPV) viral proteins E6 and E7 are considered to be pertinent to HPV-related carcinogenesis. Hence, our aim was to estimate LSCC and HPSCC for HR-HPV DNA, p16, and E6/E7 oncoprotein status by using molecular virology and immunohistochemistry methods. The prevalence of HPV16 infection was 22/41 (53.7%) and 20/31 (64.5%) for LSCC and HPSCC, accordingly. The majority of HPV16+ tumor samples were stage III or IV. In most samples, the presence of either HPV16 E6 or HPV16 E7 viral protein in dysplastic or tumor cells was confirmed using immunohistochemistry. Our results suggest a high prevalence of HPV16 as a primary HR-HPV type in LSCC and HPSCC. The lack of HPV E6/E7 oncoproteins in some tumor samples may suggest either the absence of viral integration or the presence of other mechanisms of tumorigenesis. The utilization of p16 IHC as a surrogate marker of HR-HPV infection is impractical in LSCC and HPSCC.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic pathogen. In 2012, the infectious outbreak caused by MERS-CoV in Saudi Arabia has spread to more than 1600 patients in 26 countries, resulting in over 600 deaths.Without a travel history, few clinical and radiological features can reliably differentiate MERS from SARS. But in real world, comparing with SARS, MERS presents more vaguely defined epidemiology, more severe symptoms, and higher case fatality rate. In this review, we summarize the recent findings in the field of MERS-CoV, especially its molecular virology, interspecies mechanisms, clinical features, antiviral therapies, and the further investigation into this disease. As a newly emerging virus, many questions are not fully answered, including the exact mode of transmission chain, geographical distribution, and animal origins. Furthermore, a new protocol needs to be launched to rapidly evaluate the effects of unproven antiviral drugs and vaccine to fasten the clinical application of new drugs.

Institute of Molecular Virology, Ulm University Medical Centre, Meyerhofstrasse 1, 89081 Ulm, Germany *Author for correspondence: jan.muench@uni-ulm.de

The 5'-cap structures of eukaryotic mRNAs are important for RNA stability, pre-mRNA splicing, mRNA export, and protein translation. Many viruses have evolved mechanisms for generating their own cap structures with methylation at the N7 position of the capped guanine and the ribose 2'-Oposition of the first nucleotide, which help viral RNAs escape recognition by the host innate immune system. The RNA genomes of coronavirus were identified to have 5'-caps in the early 1980s. However, for decades the RNA capping mechanisms of coronaviruses remained unknown. Since 2003, the outbreak of severe acute respiratory syndrome coronavirus has drawn increased attention and stimulated numerous studies on the molecular virology of coronaviruses. Here, we review the current understanding of the mechanisms adopted by coronaviruses to produce the 5'-cap structure and methylation modification of viral genomic RNAs.

Infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the Coronaviridae family and is a positive-sense single-stranded RNA virus originating from Wuhan, China, was declared a global public health emergency on 11 March 2020. SARS-CoV-2 infection in humans is characterized by symptoms such as fever and dyspnea accompanied by infrequent incidence of lymphopenia, gastrointestinal complications such as elevated hepatic aminotransferases, and diarrhea. Originating in bats, the SARS-CoV-2 virus has been transmitted to humans likely via an intermediate host that is yet to be discovered. Owing to the absence of any vaccines or definite anti-viral drugs alongside the greater mobility of people across the globe, international and national efforts in containing and treating SARS-CoV-2 infection are experiencing severe difficulties. In this review, we have provided a picture of SARS-CoV-2 epidemiological characteristics, the clinical symptoms experienced by patients of varying age groups, the molecular virology of SARS-CoV-2, and the treatment regimens currently employed for fighting SARS-CoV-2 infection as well as their outcomes.

The 30th meeting of the Brazilian Society for Virology (SBV) was held, for the first time in its 30 years of existence, in Cuiabá, the capital of Mato Grosso State, Central Western Brazil, a tropical region between the three richest biomes in the world: Amazon Florest, Cerrado and Pantanal. In recent years, the field of virology has been built in the State. The aim of this report is to support participants and virologists to receive the most up-to-date information about the meeting, which occurred from 16 to 19 October 2019. National and international speakers gave SBV the opportunity to learn about their experience on their virology fields, sharing recent scientific findings, compiling conferences, round table presentations and work presentations in oral and poster sessions. The meeting held over 300 attendants, who were also involved on oral and poster presentations, showing a great variety of recent unpublished studies on environmental, basic, animal, human, plant and invertebrate virology. In addition, SBV offered the Helio Gelli Pereira award for the best research studies in each field presented during the meeting. The 30th meeting of SBV was very productive and has also encouraged scientific partnership and collaboration among virologists worldwide.

The generation of wild-type, mutant, and reassortant influenza viruses from viral cDNAs (reverse genetics) is now a basic molecular virology technique in many influenza virus laboratories. Here, I describe the original RNA polymerase I reverse genetics system and the modifications that have been developed in past years. Together, these technologies have made possible many advances in basic and applied influenza virology that would not have been otherwise attainable, including the revival and study of extinct influenza viruses, the rapid characterization of emerging influenza viruses, the generation of conventional influenza vaccines, and the development of novel influenza vaccines.

Since 2016, hepatic rupture haemorrhage syndrome (HRHS) appeared in chickens of China and caused huge economic loss. To assess the infection status of the avian hepatitis E virus (HEV) and co-infected viruses, including avian leukosis virus (ALV), reticuloendotheliosis virus (REV), fowl adenovirus (FAdV), and chicken infectious anaemia virus (CIAV), in farms with HRHS, 180 liver samples were collected from 24 farms in different provinces and detected by strict molecular virology methods. Results showed that the positive rates of HEV, ALV, REV, FAdV, and CIAV were 74.44%, 20.00%, 27.78%, 31.11%, and 12.22%, respectively, whereas there are also 112 samples with co-infection, for a rate of 58%. Meanwhile, the positive rate of HEV decreased gradually with age; the lowest positive rate of ALV (5.76%) and REV (19.23%) appeared in 25-35 weeks age, during which the positive rate of CIAV was the highest (19.23%); the positive rate of HEV in layers (64.00%) was lower than that of broilers (83.33%), but the positive rates of ALV (38.46%) and CIAV (15.38%) in layers were higher than that of broilers (5.88%, 9.80%); the positive rates of HEV (75.88%) and CIAV (15.60%) in parental generation (PG) were higher than that of commodity generation (CG, 64.10%, 0.00%), whereas the positive rate of ALV showed inverse relationship (PG: 14.89%; CG: 38.46%). Additionally, phylogenetic analysis showed that all the avian HEV identified this study belong to a novel genotype, and found the close relationship between the wild strains (REV and CIAV) and corresponding isolates from contaminated vaccine. The data presented in this report will enhance the current understanding of the epidemiology characteristics in farms with HRHS in China.

Annual seasonal influenza vaccines are composed of two influenza A strains representing the H1N1 and H3N2 subtypes, and two influenza B strains representing the Victoria and Yamagata lineages. Strains from these Influenza A and Influenza B viruses currently co-circulate in humans. Of these, strains associated with the H3N2 subtype are affiliated with severe influenza seasons. H3N2 influenza viruses pre-dominated during 3 of the last 5 quite severe influenza seasons. During the 2016/2017 flu season, the H3N2 component of the influenza vaccine exhibited a poor protective efficacy (∼28-42%) against preventing infection of co-circulating strains. Since their introduction to the human population in 1968, H3N2 Influenza viruses have rapidly evolved both genetically and antigenically in an attempt to escape host immune pressures. As a result, these viruses have added numerous N-linked glycans to the viral hemagglutinin (HA), increased the overall net charge of the HA molecule, changed their preferences in receptor binding, and altered the ability of neuraminidase (NA) to agglutinate red blood cells prior to host entry. Over time, these adaptations have made characterizing these viruses increasingly difficult. This review investigates these recent changes in modern H3N2 influenza viruses and explores the methods that researchers are currently developing in order to study these viruses.

Chikungunya virus (CHIKV) was discovered more than six decades ago, but has remained poorly investigated. However, after a recent outbreak of CHIK fever in both hemispheres and viral adaptation to new species of mosquitoes, it has attracted a lot of attention. The currently available experimental data suggest that molecular mechanisms of CHIKV replication in vertebrate and mosquito cells are similar to those of other New and Old World alphaviruses. However, this virus exhibits a number of unique characteristics that distinguish it from the other, better studied members of the alphavirus genus. This review is an attempt to summarize the data accumulated thus far regarding the molecular mechanisms of alphavirus RNA replication and interaction with host cells. Emphasis was placed on demonstrating the distinct features of CHIKV in utilizing host factors to build replication complexes and modify the intracellular environment for efficient viral replication and inhibition of the innate immune response. The available data suggest that our knowledge about alphavirus replication contains numerous gaps that potentially hamper the development of new therapeutic means against CHIKV and other pathogenic alphaviruses.