Broad Impact of Exchange Protein Directly Activated by cAMP 2 (EPAC2) on Respiratory Viral Infections

The recently discovered exchange protein directly activated by cAMP (EPAC), compared with protein kinase A (PKA), is a fairly new family of cAMP effectors. Soon after the discovery, EPAC has shown its significance in many diseases including its emerging role in infectious diseases. In a recent study, we demonstrated that EPAC, but not PKA, is a promising therapeutic target to regulate respiratory syncytial virus (RSV) replication and its associated inflammation. In mammals, there are two isoforms of EPAC—EPAC1 and EPAC2. Unlike other viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) and Ebola virus, which use EPAC1 to regulate viral replication, RSV uses EPAC2 to control its replication and associated cytokine/chemokine responses. To determine whether EPAC2 protein has a broad impact on other respiratory viral infections, we used an EPAC2-specific inhibitor, MAY0132, to examine the functions of EPAC2 in human metapneumovirus (HMPV) and adenovirus (AdV) infections. HMPV is a negative-sense single-stranded RNA virus belonging to the family Pneumoviridae, which also includes RSV, while AdV is a double-stranded DNA virus. Treatment with an EPAC1-specific inhibitor was also included to investigate the impact of EPAC1 on these two viruses. We found that the replication of HMPV, AdV, and RSV and the viral-induced immune mediators are significantly impaired by MAY0132, while an EPAC1-specific inhibitor, CE3F4, does not impact or slightly impacts, demonstrating that EPAC2 could serve as a novel common therapeutic target to control these viruses, all of which do not have effective treatment and prevention strategies.     

Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus

Hepatitis C virus (HCV), the major causative agent of chronic and sporadic non-A, non-B hepatitis worldwide, is a distinct member of the Flaviviridae virus family. These viruses have in common a plus-strand RNA genome that is replicated in the cytoplasm of the infected cell via minus-strand RNA intermediates. Owing to the lack of reliable cell culture systems and convenient animal models for HCV, the mechanisms governing RNA replication are not known. As a first step towards the development of appropriate in vitro systems, we expressed the NS5B RNA-dependent RNA polymerase (RdRp) in insect cells, purified the protein to near homogeneity and studied its biochemical properties. It is a primer- and RNA template-dependent RNA polymerase able to copy long heteropolymeric templates without additional viral or cellular cofactors. We determined the optimal reaction parameters, the kinetic constants and the substrate specificity of the enzyme, which turned out to be similar to those described for the 3D polymerase of poliovirus. By analysing a series of nucleosidic and non-nucleosidic compounds for their effect on RdRp activity, we found that ribavirin triphosphates have no inhibitory effect, providing direct experimental proof that the therapeutic effect observed in patients is not related to a direct inhibition of the viral polymerase. Finally, mutation analysis was performed to map the minimal NS5B sequence required for enzymatic activity and to identify the 'classical' polymerase motifs important for template and NTP binding and catalysis.     

Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP,Mutations, and the CMV 2b Silencing Suppressor

Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species.     

Incidence and morbidity of human metapneumovirus and other community‐acquired respiratory viruses in lung transplant recipients

A. Weinberg, D.M. Lyu, S. Li, J. Marquesen, M.R. Zamora. Incidence and morbidity of human metapneumovirus and other community‐acquired respiratory viruses in lung transplant recipients
Transpl Infect Dis 2010: 12: 330–335. All rights reserved. Abstract: To determine the role of human metapneumovirus (HMPV) in respiratory tract infections (RTIs) of lung transplant recipients, 60 patients were prospectively enrolled in this study spanning from September 2005 to November 2007. Community‐acquired respiratory viruses (CARVs) were identified by polymerase chain reaction and tissue culture in respiratory secretions. Of 112 RTIs, 51 were associated with ≥1 CARV, including 7 HMPV, 13 respiratory syncytial virus (RSV), 19 parainfluenza virus 1, 2, or 3 (PIV), 16 influenza A or B (FLU), and 3 human rhinoviruses (HRV). Sixteen CARV‐RTIs had multiple pathogens. While the standard protocol was to admit all paramyxoviral RTIs for inhaled ribavirin, 16% CARV‐RTIs required hospitalization because of the severity of their respiratory compromise, including 25% of HPMV‐single‐agent RTI, 38% of RSV single‐agent RTI, 10% of PIV‐single‐agent RTI, and 19% of multiple‐agent RTIs. None of those with non‐CARV RTIs required hospitalization. The incidence of clinically diagnosed acute graft rejection in the first 2 months after an RTI varied from 0 for single‐agent HRV to 88% for single‐agent RSV (25% for single‐agent HMPV). A new diagnosis of chronic graft rejection in the first year after an RTI was made in approximately 25% of the RTIs and did not significantly vary with the etiologic agent. No deaths occurred during this study. In conclusion, HMPV was associated with 6% of the RTIs in lung transplant recipients and its morbidity was similar to the average moribidity of CARVs.     

Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus,Respiratory Syncytial Virus and Influenza A Virus

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca²⁺ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.     

Probenecid Inhibits Respiratory Syncytial Virus (RSV) Replication

RNA viruses like SARS-CoV-2, influenza virus, and respiratory syncytial virus (RSV) are dependent on host genes for replication. We investigated if probenecid, an FDA-approved and safe urate-lowering drug that inhibits organic anion transporters (OATs) has prophylactic or therapeutic efficacy to inhibit RSV replication in three epithelial cell lines used in RSV studies, i.e., Vero E6 cells, HEp-2 cells, and in primary normal human bronchoepithelial (NHBE) cells, and in BALB/c mice. The studies showed that nanomolar concentrations of all probenecid regimens prevent RSV strain A and B replication in vitro and RSV strain A in vivo, representing a potential prophylactic and chemotherapeutic for RSV.     

The N-Terminal α-Helix of Potato Virus X-Encoded RNA-Dependent RNA Polymerase Is Required for Membrane Association and Multimerization

Positive-sense single-stranded RNA viruses replicate in virus-induced membranous organelles for maximum efficiency and immune escaping. The replication of potato virus X (PVX) takes place on the endoplasmic reticulum (ER); however, how PVX-encoded RNA-dependent RNA polymerase (RdRp) is associated with the ER is still unknown. A proline-kinked amphipathic α-helix was recently found in the MET domain of RdRp. In this study, we further illustrate that the first α-helix of the MET domain is also required for ER association. Moreover, we found that the MET domain forms multimers on ER and the first α-helix is essential for multimerization. These results suggest that the RdRp of PVX adopts more than one hydrophobic motif for membrane association and for multimerization.     

Repurposing Probenecid to Inhibit SARS-CoV-2, Influenza Virus,and Respiratory Syncytial Virus (RSV) Replication

Viral replication and transmissibility are the principal causes of endemic and pandemic disease threats. There remains a need for broad-spectrum antiviral agents. The most common respiratory viruses are endemic agents such as coronaviruses, respiratory syncytial viruses, and influenza viruses. Although vaccines are available for SARS-CoV-2 and some influenza viruses, there is a paucity of effective antiviral drugs, while for RSV there is no vaccine available, and therapeutic treatments are very limited. We have previously shown that probenecid is safe and effective in limiting influenza A virus replication and SARS-CoV-2 replication, along with strong evidence showing inhibition of RSV replication in vitro and in vivo. This review article will describe the antiviral activity profile of probenecid against these three viruses.     

Enhancing the Antiviral Potency of Nucleobases for Potential Broad-Spectrum Antiviral Therapies

Broad-spectrum antiviral therapies hold promise as a first-line defense against emerging viruses by blunting illness severity and spread until vaccines and virus-specific antivirals are developed. The nucleobase favipiravir, often discussed as a broad-spectrum inhibitor, was not effective in recent clinical trials involving patients infected with Ebola virus or SARS-CoV-2. A drawback of favipiravir use is its rapid clearance before conversion to its active nucleoside-5′-triphosphate form. In this work, we report a synergistic reduction of flavivirus (dengue, Zika), orthomyxovirus (influenza A), and coronavirus (HCoV-OC43 and SARS-CoV-2) replication when the nucleobases favipiravir or T-1105 were combined with the antimetabolite 6-methylmercaptopurine riboside (6MMPr). The 6MMPr/T-1105 combination increased the C-U and G-A mutation frequency compared to treatment with T-1105 or 6MMPr alone. A further analysis revealed that the 6MMPr/T-1105 co-treatment reduced cellular purine nucleotide triphosphate synthesis and increased conversion of the antiviral nucleobase to its nucleoside-5′-monophosphate, -diphosphate, and -triphosphate forms. The 6MMPr co-treatment specifically increased production of the active antiviral form of the nucleobases (but not corresponding nucleosides) while also reducing levels of competing cellular NTPs to produce the synergistic effect. This in-depth work establishes a foundation for development of small molecules as possible co-treatments with nucleobases like favipiravir in response to emerging RNA virus infections.     

Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase.

The association of host proteins with viral RNA replication proteins has been reported for a number of (+)-strand RNA viruses. However, little is known about the identity or function of these host proteins in viral replication. In this paper we report the characterization of a host protein associated with the RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley. A host protein was specifically and proportionally enriched with BMV RdRp activity through several purification steps. This RdRp-associated host protein reacted with an antiserum prepared against wheat germ eukaryotic translation initiation factor 3 (eIF-3). The RdRp-associated host protein, the p41 subunit of wheat germ eIF-3, and an antigenically related protein from rabbit reticulocyte lysates were all found to bind with high affinity and specificity to BMV-encoded protein 2a, which is involved in viral RNA replication. Moreover, addition of wheat germ eIF-3 or the p41 subunit from wheat germ to BMV RdRp gave a specific and reproducible 3-fold stimulation of (-)-strand RNA synthesis in vivo. These results suggest that the barley analog of eIF-3 subunit p41, or a closely related protein, associates with BMV RdRp in vivo and is involved in BMV RNA replication. This observation and the established role of translation factors in bacteriophage Q beta RdRp suggest that association with translation factors may be a general feature of RNA replication by (+)-strand RNA viruses.     

Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis

The 3′untranslated region (3′UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3′UTR and vC/b3′UTR, with 3′UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3′UTR and vC/b3′UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3′UTR or vC/b3′UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3′UTR_P162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5B_P162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.     

Metagenomic Analysis of Marigold: Mixed Infection Including Two New Viruses

Marigold plants with symptoms of mosaic, crinkle, leaf curl and necrosis were observed and small RNA and ribo-depleted total RNA deep sequencing were conducted to identify the associated viruses. Broad bean wilt virus 2, cucumber mosaic virus, turnip mosaic virus, a new potyvirus tentatively named marigold mosaic virus (MMV) and a new partitivirus named as marigold cryptic virus (MCV) were finally identified. Complete genome sequence analysis showed MMV was 9811 nt in length, encoding a large polyprotein with highest aa sequence identity (57%) with the putative potyvirus polygonatumkingianum virus 1. Phylogenetic analysis with the definite potyviruses based on the polyprotein sequence showed MMV clustered closest to plum pox virus. The complete genome of MCV comprised of dsRNA1 (1583 bp) and dsRNA2 (1459 bp), encoding the RNA-dependent RNA polymerase (RdRp), and coat protein (CP), respectively. MCV RdRp shared the highest (75.7%) aa sequence identity with the unclassified partitivirus ambrosia cryptic virus 2, and 59.0%, 57.1%, 56.1%, 54.5% and 33.7% with the corresponding region of the definite delta-partitiviruses, pepper cryptic virus 2, beet cryptic virus 3, beet cryptic virus 2, pepper cryptic virus 1 and fig cryptic virus, respectively. Phylogenetic analysis based on the RdRp aa sequence showed MCV clustered into the delta-partitivirus group. These findings enriched our knowledge of viruses infecting marigold, but the association of the observed symptom and the identified viruses and the biological characterization of the new viruses should be further investigated.     

Novel Fusari- and Toti-like Viruses,with Probable Different Origins,in the Plant Pathogenic Oomycete Globisporangium ultimum

To further classify the oomycete viruses that have been discovered in recent years, we investigated virus infection in the plant-parasitic oomycete Globisporangium ultimum in Japan. Double-stranded RNA detection, high-throughput sequencing, and RT-PCR revealed that the G. ultimum isolate UOP226 contained two viruses related to fusarivirus and totivirus, named Pythium ultimum RNA virus 1 (PuRV1) and Pythium ultimum RNA virus 2 (PuRV2), respectively. Phylogenetic analysis of the deduced amino acid sequence of the RNA-dependent RNA polymerase (RdRp) showed that fusari-like PuRV1 belonged to a different phylogenetic group than Plasmopara viticola lesion-associated fusari virus (PvlaFV) 1–3 from oomycete Plasmopara viticola. Codon usage bias of the PuRV1 RdRp gene was more similar to those of fungi than Globisporangium and Phytophthora, suggesting that the PuRV1 ancestor horizontally transmitted to G. ultimum ancestor from fungi. Phylogenetic analysis of the deduced amino acid sequence of the RdRp of toti-like PuRV2 showed a monophyletic group with the other toti-like oomycete viruses from Globisporangium, Phytophthora, and Pl. viticola. However, the nucleotide sequences of toti-like oomycete viruses were not so homologous, suggesting the possibility of convergent evolution of toti-like oomycete viruses.     

Influence of Ribavirin on Mumps Virus Population Diversity

Frequent mumps outbreaks in vaccinated populations and the occurrence of neurological complications (e.g., aseptic meningitis or encephalitis) in patients with mumps indicate the need for the development of more efficient vaccines as well as specific antiviral therapies. RNA viruses are genetically highly heterogeneous populations that exist on the edge of an error threshold, such that additional increases in mutational burden can lead to extinction of the virus population. Deliberate modulation of their natural mutation rate is being exploited as an antiviral strategy and a possibility for rational vaccine design. The aim of this study was to examine the ability of ribavirin, a broad-spectrum antiviral agent, to introduce mutations in the mumps virus (MuV) genome and to investigate if resistance develops during long-term in vitro exposure to ribavirin. An increase in MuV population heterogeneity in the presence of ribavirin has been observed after one passage in cell culture, as well as a bias toward C-to-U and G-to-A transitions, which have previously been defined as ribavirin-related. At higher ribavirin concentration, MuV loses its infectivity during serial passaging and does not recover. At low ribavirin concentration, serial passaging leads to a more significant increase in population diversity and a stronger bias towards ribavirin-related transitions, independently of viral strain or cell culture. In these conditions, the virus retains its initial growth capacity, without development of resistance at a whole-virus population level.     

人类偏肺病毒与呼吸道合胞病毒呼吸道感染的临床研究

目的 了解福州地区人类偏肺病毒(HMPV)感染情况,比较HMPV与呼吸道合胞病毒(RSV)引起呼吸道感染的临床特征及流行特点.方法 采集2005年至2007年连续两个冬春季节153份福建省立医院就诊呼吸道感染患者痰标本或咽拭子标本,RT-PCR和套式RT-PCR分别检测RSV和HMPV,部分阳性PCR产物测序,DNAMAN软件分析;结合临床资料,比较两种病毒所引起的呼吸道感染临床症状、体征和流行特点.结果 153份鼻咽分泌物标本中,32份HMPV阳性,阳性率为20.9%;26份RSV阳性,阳性率为17.0%,8份HMPV和RSV均阳性.随机抽取3份标本,HMPV核苷酸序列一致,登载NCBI基因库(序列号DQ887758),基因进化树分析为单一基因型,属A基因型,但发生部分变异.2005年至2006年冬春HMPV阳性26份,阳性率为26.7%,2006年至2007年冬春HMPV阳性6份,阳性率为10.7%,而RSV检出情况与HMPV相反.儿童RSV感染平均年龄为(2.65±2.65)岁,HMPV为(4.58±3.35)岁.两种病毒引起呼吸道感染症状均以咳嗽、咽痛,发热为主.结论 HMPV与RSV均是福州地区冬春季节呼吸道病毒感染的主要病原体,两者可合并感染;HMPV主要感染年龄较大儿童,HMPV与RSV的临床特征相似.本研究期间福州地区发现的HMPV为单一基因型.