Characterization of a mammalian orthoreovirus isolated from the large flying fox, Pteropus vampyrus, in Indonesia.

Fruit bats serve as an important reservoir for many zoonotic pathogens, including Nipah virus, Hendra virus, Marburg virus and Lyssavirus. To gain a deeper insight into the virological characteristics, pathogenicity and zoonotic potential of bat-borne viruses, recovery of infectious viruses from field samples is important. Here, we report the isolation and characterization of a mammalian orthoreovirus (MRV) from a large flying fox (Pteropus vampyrus) in Indonesia, which is the first detection of MRV in Southeast Asia. MRV was recovered from faecal samples of three different P. vampyrus in Central Java. Nucleotide sequence analysis revealed that the genome of the three MRV isolates shared more than 99% nucleotide sequence identity. We tentatively named one isolated strain as MRV12-52 for further analysis and characterization. Among 10 genome segments, MRV12-52 S1 and S4, which encode the cell-attachment protein and outer capsid protein, had 93.6 and 95.1% nucleotide sequence identities with known MRV strains, respectively. Meanwhile, the remaining genome segments of MRV12-52 were divergent with 72.9-80.7 % nucleotide sequence identities. Based on the nucleotide sequence of the S1 segment, MRV12-52 was grouped into serotype 2, and phylogenetic analysis demonstrated evidence of past reassortment events. In vitro characterization of MRV12-52 showed that the virus efficiently replicated in BHK-21, HEK293T and A549 cells. In addition, experimental infection of laboratory mice with MRV12-52 caused severe pneumonia with 75% mortality. This study highlights the presence of pathogenic MRV in Indonesia, which could serve as a potential animal and public health concern.

The effective factors in human-specific tropism and viral pathogenicity in orthopoxviruses.

The orthopoxvirus (OPV) genus includes several species that infect humans, including variola, monkeypox, vaccinia, and cowpox. Variola and monkeypox are often life-threatening diseases, while vaccinia and cowpox are usually associated with local lesions. The epidemic potential for OPVs may be lower than respiratory-borne viruses or RNA viruses. However, OPVs are notable for their spread and distribution in different environments and among different hosts. The emergence or re-emergence of OPVs in the human population can also occur in wild or domestic animals as intermediate hosts. More effective and safer vaccines for poxvirus can be developed by understanding how immunity is regulated in poxvirus and vaccines for DNA viruses. Downstream events in cells affected by the virus are regulated functionally by a series of characteristics that are affected by host cell interactions and responses of cells against viral infections, including the interferon pathway and apoptosis. Furthermore, infection outcome is greatly influenced by the distinct selection of host-range and immune-modulatory genes that confer the potential for pathogenesis and host-to-host transmission and the distinct host-range properties of each immune-modulatory gene. The present study reviewed the effective factors in human-restricted tropism and virus pathogenicity in OPVs.

Limited environmental stability of infectious porcine endogenous retrovirus type C; Usage of reverse transcriptase in combination with viral RNA as markers for infectious virus.

INTRODUCTION: Porcine endogenous retroviruses (PERVs) are an integral part of the pig genome with infectious potential, as shown in vitro. HYPOTHESIS/GAP STATEMENT: In view of nonclinical and clinical xenotransplantation, data are essential that give an insight into viral pathogenicity. This includes PERV's environmental stability and environmental risk. AIM: We analyzed two ecotropic PERV-C (PERV-C[1312] and -[5683]), monitoring cell-free culture supernatants of infected ST-IOWA cells at various time intervals at room temperature (22°C +/-1°C). The virus was stored in the presence or absence of sterile wood litter, as used for large animal husbandry. This approach was set to determine the environmental stability of exogenous PERV-C at defined conditions for the first time. METHODOLOGY: Reverse transcriptase (RT) activity and viral RNA were monitored for up to 57 days and remaining infectivity of supernatant without wood litter was tested from day 7 onwards on naïve ST-IOWA cells. RESULTS: Results show that viral RNA decreases but remains detectable over the whole observation period, whereas RT activity showed 83%-96% reduction from day 7 on. This effect was stronger in the presence of wood litter and fresh harvested virus was more stable than frozen virus stocks. Even under these optimal conditions, no infectivity was shown for viral RNA-positive and RT-reduced supernatant harvested at day 7. CONCLUSION: The results confirm that PERV-C is less stable and the reduction of RT activity is accompanied by reduced infectivity, independently of existing viral RNA. The combination of both RT and viral RNA measurement is a suitable method to differentiate infectious PERV-C.

Viral long non-coding RNA regulates virus life-cycle and pathogenicity.

Viral infection is still a serious global health problem that kills hundreds of thousands of people annually. Understanding the mechanism by which virus replicates, packages, and infects the host cells can provide new strategies to control viral infection. Long non-coding RNAs (lncRNAs) have been identified as critical regulators involved in viral infection process and antiviral response. A lot of host lncRNAs have been identified and shown to be involved in antiviral immune response during viral infection. However, our knowledge about lncRNAs expressed by viruses is still at its infancy. LncRNAs expressed by viruses are involved in the whole viral life cycle, including promoting genome replication, regulating gene expression, involvement in genome packaging, assembling new viruses and releasing virions to the host cells. Furthermore, they enhance the pathogenicity of viral infections by down-regulating the host cell's antiviral immune response and maintain the viral latency through a refined procedure of genome integration. This review focuses on the regulatory roles of viral lncRNA in the life-cycle and pathogenicity of viruses. It gives an insight into the viral lncRNAs that can be utilized as therapeutic targets against viral diseases, and future researches aimed to identify and explore new viral lncRNAs and the mechanisms of their involvement in viral infection is encouraged.

Clinical Importance of Placental Testing among Suspected Cases of Congenital Zika Syndrome.

Contemporaneous Zika virus (ZIKV) strains can cause congenital Zika syndrome (CZS). Current ZIKV clinical laboratory testing strategies are limited and include IgM serology (which may wane 12 weeks after initial exposure) and nucleic acid testing (NAT) of maternal serum, urine, and placenta for (+) strand ZIKV RNA (which is often transient). The objectives of this study were to determine if use of additional molecular tools, such as quantitative PCR and microscopy, would add to the diagnostic value of current standard placental ZIKV testing in cases with maternal endemic exposure and indeterminate testing. ZIKV RNA was quantified from dissected sections of placental villi, chorioamnion sections, and full cross-sections of umbilical cord in all cases examined. Quantitation with high-resolution automated electrophoresis determined relative amounts of precisely verified ZIKV (74-nt amplicons). In order to localize and visualize stable and actively replicating placental ZIKV in situ, labeling of flaviviridae glycoprotein, RNA ISH against both (+) and (⁻) ZIKV-specific ssRNA strands, and independent histologic examination for significant pathologic changes were employed. We demonstrate that the use of these molecular tools added to the diagnostic value of placental ZIKV testing among suspected cases of congenital Zika syndrome with poorly ascribed maternal endemic exposure.

Reverse genetic approaches allowing the characterization of the rabies virus street strain belonging to the SEA4 subclade.

Rabies virus (RABV) is the causative agent of rabies, a lethal neurological disease in mammals. RABV strains can be classified into fixed strains (laboratory strains) and street strains (field/clinical strains), which have different properties including cell tropism and neuroinvasiveness. RABV Toyohashi strain is a street strain isolated in Japan from an imported case which had been bitten by rabid dog in the Philippines. In order to facilitate molecular studies of RABV, we established a reverse genetics (RG) system for the study of the Toyohashi strain. The recombinant virus was obtained from a cDNA clone of Toyohashi strain and exhibited similar growth efficiency as the original virus in cultured cell lines. Both the original and recombinant strains showed similar pathogenicity with high neuroinvasiveness in mice, and the infected mice developed a long and inconsistent incubation period, which is characteristic of street strains. We also generated a recombinant Toyohashi strain expressing viral phosphoprotein (P protein) fused with the fluorescent protein mCherry, and tracked the intracellular dynamics of the viral P protein using live-cell imaging. The presented reverse genetics system for Toyohashi strain will be a useful tool to explore the fundamental molecular mechanisms of the replication of RABV street strains.

A Review of miRNA Regulation in Japanese Encephalitis (JEV) Virus Infection.

Japanese encephalitis (JE) is a mosquito-borne disease that causes neuronal damage and inflammation of microglia, and in severe cases, it can be fatal. JE infection can resist cellular immune responses and survive in host cells. Japanese encephalitis virus (JEV) infects macrophages and peripheral blood lymphocytes. In addition to regulating biological signaling pathways, microRNAs in cells also influence virus-host interactions. Under certain circumstances, viruses can change microRNA production. These changes affect the replication and spread of the virus. Host miRNAs can contain viral pathogenicity by downregulating the antiviral immune response pathways. Simultaneous profiling of miRNA and messenger RNA (mRNA) could help us detect pathogenic factors, and dual RNA detection is possible. This work highlights important miRNAs involved in human JE infection. In this study, we have shown the important miRNAs that play significant roles in JEV infection. We found that during JEV infection, miRNA-155, miR-29b, miRNA-15b, miR-146a, miRNA-125b-5p, miRNA-30la, miR-19b-3p, and miR-124, cause upregulation of human genes whereas miRNA-432, miRNA-370, microRNA-33a-5p, and miRNA-466d-3p are responsible for downregulation of human genes respectively. Further, these miRNAs are also responsible for the inflammatory effects. Although several other miRNAs critical to the JEV life cycle are yet unknown, there is currently no evidence for the role of miRNAs in persistence.

Approaches to pandemic prevention - the chromatin vaccine.

Developing effective vaccines against viral infections have significant impacts on development, prosperity and well-being of human populations. Thus, successful vaccines such as smallpox and polio vaccines, have promoted global societal well-being. In contrast, ineffective vaccines may fuel arguments that retard scientific progress. We aim to stimulate a multilevel discussion on how to develop effective vaccines against recent and future pandemics by focusing on acquired immunodeficiency syndrome (AIDS), coronavirus disease (COVID) and other viral infections. We appeal to harnessing recent achievements in this field specifically towards a cure for current pandemics and prevention of the next pandemics. Among these, we propose to apply the HIV DNA in chromatin format - an end product of aborted HIV integration in episomal forms, i.e., the chromatin vaccines (cVacc), to elicit the epigenetic silencing and memory that prevent viral replication and infection.

Comparison of the Pathogenicity of SARS-CoV-2 Delta and Omicron Variants by Analyzing the Expression Patterns of Immune Response Genes in K18-hACE2 Transgenic Mice.

BACKGROUND: The recently emerged variants of the severe acute respiratory coronavirus 2 (SARS-CoV-2) pose a threat to public health. Understanding the pathogenicity of these variants is a salient factor in the development of effective SARS-CoV-2 therapeutics. This study aimed to compare the expression patterns of genes involved in immune responses in K18-hACE2 mice infected with the wild-type, Delta, and Omicron SARS-CoV-2 variants. METHODS: K18-hACE2 mice were intranasally infected with either wild-type (B.1), Delta (B.1.617.2), or Omicron (B.1.1.529) variants. On day 6 post-infection, lung, brain, and kidney tissues were collected from each variant-infected group. The mRNA expression levels of 39 immune response genes in all three groups were compared by RT-qPCR. Viral titers were measured using the median tissue culture infectious dose (TCID50) assay and expressed as Log10 TCID50/0.1 g. The statistical significance of the differences in gene expression was determined by one-way analysis of variance (ANOVA) (alpha = 0.05). RESULTS: The expression of toll-like receptors (TLRs) was upregulated in the lung and brain tissues of the wild-type- and Delta-infected groups but not in those of the Omicron-infected group. The highest expression of cytokines, including interleukin (IL)-1α, IL-1ß, IL-17α, interferon, and tumor necrosis factors, was observed in the lungs of mice infected with the wild-type variant. Additionally, CCL4, CCL11, CXCL9, and CXCL10 were upregulated (>3-fold) in wild-type-infected mice, with markedly higher expressions in the brain than in the lungs. Most of the apoptotic factors were mainly expressed in the brain tissues of Omicron-infected mice (caspase 8, caspase 9, p53, Bax, Bak, BCL-2, and Bcl-XL), whereas neither the lung nor kidney showed more than 3-fold upregulation of these apoptotic factors. CONCLUSIONS: Collectively, our findings revealed that the wild-type SARS-CoV-2 variant exhibited the highest pathogenicity, followed by the Delta variant, then the Omicron variant.

Isolation and characterization of an orthoreovirus from Indonesian fruit bats.

Nelson Bay orthoreovirus (NBV) is an emerging bat-borne virus and causes respiratory tract infections in humans sporadically. Over the last two decades, several strains genetically related to NBV were isolated from humans and various bat species, predominantly in Southeast Asia (SEA), suggesting a high prevalence of the NBV species in this region. In this study, an orthoreovirus (ORV) belonging to the NBV species was isolated from Indonesian fruit bats' feces, tentatively named Paguyaman orthoreovirus (PgORV). Serological studies revealed that 81.2% (108/133) of Indonesian fruit bats sera had neutralizing antibodies against PgORV. Whole-genome sequencing and phylogenetic analysis of PgORV suggested the occurrence of past reassortments with other NBV strains isolated in SEA, indicating the dispersal and circulation of NBV species among bats in this region. Intranasal PgORV inoculation of laboratory mice caused severe pneumonia. Our study characterized PgORV's unique genetic background and highlighted the potential risk of PgORV-related diseases in Indonesia.

New substitutions on NS1 protein from influenza A (H1N1) virus: Bioinformatics analyses of Indian strains isolated from 2009 to 2020.

Background and Aims: Nonstructural (NS1) protein is mainly involved in virulence and replication of several viruses, including influenza virus A (H1N1); surveillance of the latter started in India in 2009. The objective of this study was to identify the new substitutions in NS1 protein from the influenza virus A (H1N1) pandemic 2009 (pdm09) strain isolated in India. Methods: The sequences of NS1 proteins from influenza A(H1N1) pdm09 strains isolated in India were obtained from publicly available databases. Multiple sequence alignment and phylogeny analyses were performed to confirm the "consistent substitutions" on NS1 protein from H1N1 (pdm09) Indian strains. Here, "consistent substitutions" were defined as the substitutions observed in all the sequences isolated in a year. Comparative analyses were performed among NS1 Indian sequences from A(H1N1) pdm09, A (H1N1) seasonal and A(H3N2) strains, and from A (H1N1) pdm09 global strains. Results: Eight substitutions were identified in the NS1 Indian sequence from the A(H1N1) pdm09 strain, two in RBD, five in ED, and one in the linker region. Three new substitutions were reported in this study at NS1 sequence positions 2, 80, and 155, which evolved within 2015-2019 and became "consistent." These new substitutions were associated with conservative paired substitutions in the alternative domains of the NS1 protein. Three paired substitutions were (i) D2E and E125D, (ii) T80A and A155T, and (iii) E55K and K131E. Conclusions: This study indicates the continuous evolution of NS1 protein from the influenza A virus. The new substitutions at positions 2 and 80 occurred in the RNA binding and eIF4GI binding domains. The D2E substitution evolved simultaneously with the E125D substitution that involved viral replication. The third new substitution at position 155 occurred in the PI3K binding domain. The possible consequences of these substitutions on host-pathogen interactions are subject to further experimental and computational verification.

Stem-Loop I of the Tembusu Virus 3'-Untranslated Region Is Responsible for Viral Host-Specific Adaptation and the Pathogenicity of the Virus in Mice.

Tembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3'-untranslated region (3'UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3'UTRs. Compared with rMM 1775, rMM-CQ3'UTR (recombinant MM 1775 virus carrying the 3'UTR of CQW1) exhibited enhanced proliferation in vitro, with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3'UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads. In contrast, rCQ-MM3'UTR showed similar growth kinetics in vitro and neurovirulence in mice compared with those of rCQW1. Then, the Stem-loop I (SLI) structure, which showed the highest variation within the 3'UTR between CQW1 and MM 1775, was further chosen for making chimeric viruses. The peak titers of rMM-CQ3'UTRSLI displayed a 15- or 4-fold increase in vitro, and the neurovirulence in mice was attenuated, compared with that of rMM 1775; rCQ-MM3'UTRSLI displayed comparable multiplication ability in vitro but was significantly attenuated in mice, in contrast with rCQW1. In conclusion, we demonstrated that the TMUV SLI structure of the 3'UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old mice, providing a new understanding of the functions of TMUV 3'UTR in viral host switching and the pathogenicity changes in mice. IMPORTANCE Mosquito-borne flaviviruses (MBFVs) constitute a large number of mosquito-transmitted viruses. The 3'-untranslated region (3'UTR) of MBFV has been suggested to be relevant to viral host-specific adaptation. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3'UTR are largely unknown. Here, using Tembusu virus (TMUV), an avian MBFV as models, we observed that the duck-derived SLI of the 3'UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3'UTR in viral host switching and pathogenicity changes in mice.

Experimental and natural infections of white-tailed sea eagles (Haliaeetus albicilla) with high pathogenicity avian influenza virus of H5 subtype.

White-tailed sea eagle (Haliaeetus albicilla), a regionally rare species of raptor, is threatened in several countries. To assess the risk of H5 high pathogenicity avian influenza (HPAI) viral infection in rare bird species, we performed experimental infections with a GS/GD96-lineage H5N6 HPAI virus of clade 2.3.4.4e in white-tailed sea eagles. Additionally, during the winter of 2020-2021 in Japan, we accidentally encountered a white-tailed sea eagle that had a fatal outcome due to natural infection with a GS/GD96-lineage H5N8 HPAI virus of clade 2.3.4.4b, allowing us to compare experimental and natural infections in the same rare raptor species. Our experiments demonstrated the susceptibility of white-tailed sea eagles to the GS/GD96-lineage H5 HPAI virus with efficient replication in systemic organs. The potential for the viruses to spread within the white-tailed sea eagle population through indirect transmission was also confirmed. Comprehensive comparisons of both viral distribution and histopathological observations between experimentally and naturally infected white-tailed sea eagles imply that viral replication in the brain is responsible for the disease severity and mortality in this species. These findings provide novel insights into the risk assessment of H5 HPAI viral infection in white-tailed sea eagles, proper diagnostic procedures, potential risks to artificially fed eagle populations and persons handling superficially healthy eagles, potential impact of intragastric infection on eagle outcomes, and possibility of severity of the disease being attributed to viral replication in the brain.

In silico characterization of mutations circulating in SARS-CoV-2 structural proteins.

SARS-CoV-2 has recently emerged as a pandemic that has caused more than 2.4 million deaths worldwide. Since the onset of infections, several full-length sequences of viral genome have been made available which have been used to gain insights into viral dynamics. We utilised a meta-data driven comparative analysis tool for sequences (Meta-CATS) algorithm to identify mutations in 829 SARS-CoV-2 genomes from around the world. The algorithm predicted sixty-one mutations among SARS-CoV-2 genomes. We observed that most of the mutations were concentrated around three protein coding genes viz nsp3 (non-structural protein 3), RdRp (RNA-directed RNA polymerase) and Nucleocapsid (N) proteins of SARS-CoV-2. We used various computational tools including normal mode analysis (NMA), C-α discrete molecular dynamics (DMD) and all-atom molecular dynamic simulations (MD) to study the effect of mutations on functionality, stability and flexibility of SARS-CoV-2 structural proteins including envelope (E), N and spike (S) proteins. PredictSNP predictor suggested that four mutations (L37H in E, R203K and P344S in N and D614G in S) out of seven were predicted to be neutral whilst the remaining ones (P13L, S197L and G204R in N) were predicted to be deleterious in nature thereby impacting protein functionality. NMA, C-α DMD and all-atom MD suggested some mutations to have stabilizing roles (P13L, S197L and R203K in N protein) where remaining ones were predicted to destabilize mutant protein. In summary, we identified significant mutations in SARS-CoV-2 genomes as well as used computational approaches to further characterize the possible effect of highly significant mutations on SARS-CoV-2 structural proteins.Communicated by Ramaswamy H. Sarma.

Understanding the Variability of Certain Biological Properties of H1N1pdm09 Influenza Viruses.

The influenza virus continually evolves because of the high mutation rate, resulting in dramatic changes in its pathogenicity and other biological properties. This study aimed to evaluate the evolution of certain essential properties, understand the connections between them, and find the molecular basis for the manifestation of these properties. To that end, 21 A(H1N1)pdm09 influenza viruses were tested for their pathogenicity and toxicity in a mouse model with a ts/non-ts phenotype manifestation and HA thermal stability. The results demonstrated that, for a strain to have high pathogenicity, it must express a toxic effect, have a non-ts phenotype, and have a thermally stable HA. The ancestor A/California/07/2009 (H1N1)pdm influenza virus expressed the non-ts phenotype, after which the cycling trend of the ts/non-ts phenotype was observed in new strains of A(H1N1)pdm09 influenza viruses, indicating that the ratio of the ts phenotype will increase in the coming years. Of the 21 tested viruses, A/South Africa/3626/2013 had the high pathogenicity in the mouse model. Sequence alignment analysis showed that this virus has three unique mutations in the polymerase complex, two of which are in the PB2 gene and one that is in the PB1 gene. Further study of these mutations might explain the distinguishing pathogenicity.

Is the human T-cell lymphotropic virus type 2 in the process of endogenization into the human genome?

Human T-cell lymphotropic virus type 2 (HTLV-2) infection has been shown to be endemic among intravenous drug users in parts of North America, Europe and Southeast Asia and in a number of Amerindian populations. Despite a 65% genetic similarity and common host humoral response, the human T-cell lymphotropic viruses type 1 (HTLV-1) and 2 display different mechanisms of host interaction and capacity for disease development. While HTLV-1 pathogenicity is well documented, HTLV-2 etiology in human disease is not clearly established. From an evolutionary point of view, its introduction and integration into the germ cell chromosomes of host species could be considered as the final stage of parasitism and evasion from host immunity. The extraordinary abundance of endogenous viral sequences in all vertebrate species genomes, including the hominid family, provides evidence of this invasion. Some of these gene sequences still retain viral characteristics and the ability to replicate and hence are potentially able to elicit responses from the innate and adaptive host immunity, which could result in beneficial or pathogenic effects. Taken together, this data may indicate that HTLV-2 is more likely to progress towards endogenization as has happened to the human endogenous retroviruses millions of years ago. Thus, this intimate association (HTLV-2/human genome) may provide protection from the immune system with better adaptation and low pathogenicity.

Long-Term Existence of SARS-CoV-2 in COVID-19 Patients: Host Immunity, Viral Virulence, and Transmissibility.

COVID-19 patients can recover with a median SARS-CoV-2 clearance of 20 days post initial symptoms (PIS). However, we observed some COVID-19 patients with existing SARS-CoV-2 for more than 50 days PIS. This study aimed to investigate the cause of viral clearance delay and the infectivity in these patients. Demographic data and clinical characteristics of 22 long-term COVID-19 patients were collected. The median age of the studied cohort was 59.83 ± 12.94 years. All patients were clinically cured after long-term SARS-CoV-2 infection ranging from 53 to 112 days PIS. Peripheral lymphocytes counts were normal. The ratios of interferon gamma (IFN-γ)-secreting cells to total CD4+ and CD8+ cells were normal as 24.68% ± 9.60% and 66.41% ± 14.87% respectively. However, the number of IFN-γ-secreting NK cells diminished (58.03% ± 11.78%). All patients presented detectable IgG, which positively correlated with mild neutralizing activity (Mean value neutralisation antibodies titers = 157.2, P = 0.05). No SARS-CoV-2 virus was isolated in Vero E6 cells inoculated with nasopharyngeal swab samples from all patients 50 days PIS, and the cytopathic effect was lacking. But one sample was positive for SARS-CoV-2 nucleic acid test in cell supernatants after two passages. Genome sequencing revealed that only three synonymous variants were identified in spike protein coding regions. In conclusion, decreased IFN-γ production by NK cells and low neutralizing antibodies might favor SARS-CoV-2 long-term existence. Further, low viral load and weak viral pathogenicity were observed in COVID-19 patients with long-term SARS-CoV-2 infection.

The Roles of prM-E Proteins in Historical and Epidemic Zika Virus-mediated Infection and Neurocytotoxicity.

The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe human congenital and neurologic disorders including microcephaly in newborns and Guillain-Barré syndrome in adults. Studies showed that the epidemic ZIKV strains are phenotypically different from the historic strains, suggesting that the epidemic ZIKV has acquired mutations associated with the altered viral pathogenicity. However, what genetic changes are responsible for the changed viral pathogenicity remains largely unknown. One of our early studies suggested that the ZIKV structural proteins contribute in part to the observed virologic differences. The objectives of this study were to compare the historic African MR766 ZIKV strain with two epidemic Brazilian strains (BR15 and ICD) for their abilities to initiate viral infection and to confer neurocytopathic effects in the human brain's SNB-19 glial cells, and further to determine which part of the ZIKV structural proteins are responsible for the observed differences. Our results show that the historic African (MR766) and epidemic Brazilian (BR15 and ICD) ZIKV strains are different in viral attachment to host neuronal cells, viral permissiveness and replication, as well as in the induction of cytopathic effects. The analysis of chimeric viruses, generated between the MR766 and BR15 molecular clones, suggests that the ZIKV E protein correlates with the viral attachment, and the C-prM region contributes to the permissiveness and ZIKV-induced cytopathic effects. The expression of adenoviruses, expressing prM and its processed protein products, shows that the prM protein and its cleaved Pr product, but not the mature M protein, induces apoptotic cell death in the SNB-19 cells. We found that the Pr region, which resides on the N-terminal side of prM protein, is responsible for prM-induced apoptotic cell death. Mutational analysis further identified four amino-acid residues that have an impact on the ability of prM to induce apoptosis. Together, the results of this study show that the difference of ZIKV-mediated viral pathogenicity, between the historic and epidemic strains, contributed in part the functions of the structural prM-E proteins.

Role of Zika Virus prM Protein in Viral Pathogenicity and Use in Vaccine Development.

Recent Zika virus (ZIKV) epidemics necessitate the urgent development of effective drugs and vaccines, which can be accelerated by an enhanced understanding of ZIKV biology. One of the ZIKV structural proteins, precursor membrane (prM), plays an important role in the assembly of mature virions through cleavage of prM into M protein. Recent studies have suggested that prM protein might be implicated in the neurovirulence of ZIKV. Most vaccines targeting ZIKV include prM as the immunogen. Here, we review progress in our understanding of ZIKV prM protein and its application in ZIKV vaccine development.

PB2 E627K or D701N substitution does not change the virulence of canine influenza virus H3N2 in mice and dogs.

Recently, canine influenza virus H3N2 (CIV H3N2) has circulated continuously in the dog populations of Asia and the United States (US). As humans have close contact with pet dogs, the circulation of CIV H3N2 is a cause for concern. Previous studies have reported that the E627K and D701N substitutions in the PB2 subunit enhanced viral pathogenicity to mammals in various influenza viruses. However, how the E627K and D701N substitutions in the PB2 subunit might affect the virulence of CIV H3N2 is unclear. Here, we constructed recombinant viruses by introducing E627K or D701N into the PB2 gene in the genetic background of A/Canine/Guangdong/02/2011H3N2 using a reverse-genetic system. The results showed that the E627K or D701N substitutions in the PB2 subunit of CIV H3N2 enhanced polymerase activity, but these substitutions did not impact viral pathogenicity in mice or beagles.