COVID-19 Pandemic: The Influence of Culture and Lessons for Collaborative Activities.

The rapid epidemiological shift from an epidemic/outbreak in Wuhan, China, to a global pandemic of COVID-19 in less than 3 months came with lessons the world's health system should learn to prepare for the future outbreaks. Since February 20, 2020, the total number of confirmed cases of COVID-19 has been increased very slowly in the countries of East Asia, including Japan, South Korea, and China, when compared with those in the Western countries. This chapter begins with an overview of the impact of COVID-19 on healthcare workers and public health facilities, followed by immediate global actions and research in response to the newly emerged pandemic. It includes an evaluation of the potential influence of culture on the implementation of different protective measures to combat the COVID-19 pandemic while at the same time offering suggestions that will make it easier for all populations to adapt protective steps against COVID-19 and other respiratory infectious diseases. Finally, the chapter provides a detailed discussion of lessons we have learned from the pandemic, leading to the conclusion that the transition from individualism to collaborative efforts is the treatment of universal pandemics.

Advancement in the development of gene/protein-based vaccines against African swine fever virus.

African swine fever (ASF) is a highly contagious acute hemorrhagic viral disease, with the mortality rate of up to 100 % in domestic pigs. In recent years, ASF outbreaks have caused huge economic losses in numerous countries and regions, especially in Asia. Therefore, there is a pressing need to develop safe and effective vaccines against infection of the causative pathogen, African swine fever virus (ASFV). ASFV contains a large genome composed of double-stranded DNA with a size of 170-194 kb, which encodes nearly 200 viral proteins. Understanding the function of these complex genes/proteins and their roles in the generation of protective immunity will help in the development of ASFV vaccines. In this article, the gene/protein-based vaccine candidate are summarized, and the structural proteins which have been previously reported to protect animals from the virus challenge were emphatically described.

HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain.

Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors.

Interactions between different generation HIV-1 fusion inhibitors and the putative mechanism underlying the synergistic anti-HIV-1 effect resulting from their combination.

We previously reported that the combinatorial use of T20 and T1144, the first and next generations of HIV fusion inhibitors, containing different functional domains resulted in synergistic anti-HIV-1 effect, but this effect diminished when T20 and T1144 were covalently linked together. To elucidate the mechanism underlying this synergistic anti-HIV-1 effect, we studied the interactions between T20 and T1144 either in a mixture state or in a covalently linked state. T20 alone in solution was largely featureless, while T1144 alone was in α-helical trimeric conformation. When mixed in solution, T20 and T1144 showed a loose and transient interaction, with a moderate 10% α-helical content increase, but this interaction was greatly enhanced in the linked state, and T20 and T1144 showed ∼100% α-helical content. These results suggested that the loose and transient interaction between T20 and T1144 may destabilize the T1144 trimer, which makes its otherwise shielded binding sites more accessible to N-terminal heptad repeat (NHR) and increases its associating rate, thus increasing its anti-HIV-1 potency against the temporarily exposed target in NHR and causing the synergistic anti-HIV-1 effect. However, the strong interaction between T20 and T1144 in the covalently linked state may shield their NHR-binding sites, resulting in reduction of the synergistic effect.

Potential cross-species transmission of highly pathogenic avian influenza H5 subtype (HPAI H5) viruses to humans calls for the development of H5-specific and universal influenza vaccines.

In recent years, highly pathogenic avian influenza H5 subtype (HPAI H5) viruses have been prevalent around the world in both avian and mammalian species, causing serious economic losses to farmers. HPAI H5 infections of zoonotic origin also pose a threat to human health. Upon evaluating the global distribution of HPAI H5 viruses from 2019 to 2022, we found that the dominant strain of HPAI H5 rapidly changed from H5N8 to H5N1. A comparison of HA sequences from human- and avian-derived HPAI H5 viruses indicated high homology within the same subtype of viruses. Moreover, amino acid residues 137A, 192I, and 193R in the receptor-binding domain of HA1 were the key mutation sites for human infection in the current HPAI H5 subtype viruses. The recent rapid transmission of H5N1 HPAI in minks may result in the further evolution of the virus in mammals, thereby causing cross-species transmission to humans in the near future. This potential cross-species transmission calls for the development of an H5-specific influenza vaccine, as well as a universal influenza vaccine able to provide protection against a broad range of influenza strains.

Natural variant R246K in hemagglutinin increased zoonotic characteristics and renal inflammation in mice infected with H9N2 influenza virus.

Considered a potential pandemic candidate, the widespread among poultry of H9N2 avian influenza viruses across Asia and North Africa pose an increasing threat to poultry and human health. The massive epidemic of H9N2 viruses has expanded the host range; however, the molecular basis and characteristic underlying the transmission to poultry and mammals remains unclear. Our previous study has proved that some natural mutations in the HA gene enhanced the binding ability of the H9N2 virus to α-2,6 SA receptors. Here, we systematically analyzed the impact of these natural mutations on zoonotic characteristics and the pathogenicity of H9N2 AIVs in poultry and mammals. Our study demonstrated that mutation R246K increased the replication in human lung epithelial cells in vitro. Mutation R246K increased the virus shedding of oropharyngeal swabs during early-stage infection in chickens. Moreover, mutation R246K displayed stronger pH stability and pathogenicity in mice. The strong renal tropism and inflammatory response may accelerate the pathogenicity. In summary, we found that natural variation R246K in HA of prevalent H9N2 in China promoted the transmissibility in chicken and accelerate the pathogenicity in mice, posing a great concern for zoonotic and pandemic emergence.

Nonenveloped Avian Reoviruses Released with Small Extracellular Vesicles Are Highly Infectious.

Vesicle-encapsulated nonenveloped viruses are a recently recognized alternate form of nonenveloped viruses that can avoid immune detection and potentially increase systemic transmission. Avian orthoreoviruses (ARVs) are the leading cause of various disease conditions among birds and poultry. However, whether ARVs use cellular vesicle trafficking routes for egress and cell-to-cell transmission is still poorly understood. We demonstrated that fusogenic ARV-infected quail cells generated small (~100 nm diameter) extracellular vesicles (EVs) that contained electron-dense material when observed by transmission electron microscope. Cryo-EM tomography indicated that these vesicles did not contain ARV virions or core particles, but the EV fractions of OptiPrep gradients did contain a small percent of the ARV virions released from cells. Western blotting of detergent-treated EVs revealed that soluble virus proteins and the fusogenic p10 FAST protein were contained within the EVs. Notably, virus particles mixed with the EVs were up to 50 times more infectious than virions alone. These results suggest that EVs and perhaps fusogenic FAST-EVs could contribute to ARV virulence.

A novel chimeric protein-based HIV-1 fusion inhibitor targeting gp41 glycoprotein with high potency and stability.

T20 (enfuvirtide, Fuzeon) is the first generation HIV-1 fusion inhibitor approved for salvage therapy of HIV-1-infected patients refractory to current antiretroviral drugs. However, its application is limited by the high cost of peptide synthesis, rapid proteolysis, and poor efficacy against emerging drug-resistant strains. Here we reported the design of a novel chimera protein-based fusion inhibitor targeting gp41, TLT35, that uses a flexible 35-mer linker to couple T20 and T1144, the first and next generation HIV-1 fusion inhibitors, respectively. TLT35, which was expressed in Escherichia coli with good yield, showed low nm activity against HIV-1-mediated cell-cell fusion and infection by laboratory-adapted HIV-1 strains (X4 or R5), including T20-resistant variants and primary HIV-1 isolates of clades A to G and group O (R5 or X4R5). TLT35 was stable in human sera and in peripheral blood mononuclear cell culture and was more resistant to proteolysis than either T20 or T1144 alone. Circular dichroism spectra showed that TLT35 folded into a thermally stable conformation with high α-helical content and T(m) value in aqueous solution. It formed a highly stable complex with gp41 N-terminal heptad repeat peptide and blocked formation of the gp41 six-helix-bundle core. These merits combined with an anticipated low production cost for expression of TLT35 in E. coli make this novel protein-based fusion inhibitor a promising candidate for further development as an anti-HIV-1 microbicide or therapeutic for the prevention and treatment of HIV-1 infection.

A bivalent recombinant protein inactivates HIV-1 by targeting the gp41 prehairpin fusion intermediate induced by CD4 D1D2 domains.

BACKGROUND: Most currently approved anti-HIV drugs (e.g., reverse transcriptase inhibitors, protease inhibitors and fusion/entry inhibitors) must act inside or on surface of the target cell to inhibit HIV infection, but none can directly inactivate virions away from cells. Although soluble CD4 (sCD4) can inactivate laboratory-adapted HIV-1 strains, it fails to reduce the viral loads in clinical trials because of its low potency against primary isolates and tendency to enhance HIV-1 infection at low concentration. Thus, it is essential to design a better HIV inactivator with improved potency for developing new anti-HIV therapeutics that can actively attack the virus in the circulation before it attaches to and enter into the target cell. RESULTS: We engineered a bivalent HIV-1 inactivator, designated 2DLT, by linking the D1D2 domain of CD4 to T1144, the next generation HIV fusion inhibitor, with a 35-mer linker. The D1D2 domain in this soluble 2DLT protein could bind to the CD4-binding site and induce the formation of the gp41 prehairpin fusion-intermediate (PFI), but showed no sCD4-mediated enhancement of HIV-1 infection. The T1144 domain in 2DLT then bound to the exposed PFI, resulting in rapid inactivation of HIV-1 virions in the absence of the target cell. Beside, 2DLT could also inhibit fusion of the virus with the target cell if the virion escapes the first attack of 2DLT. CONCLUSION: This bivalent molecule can serve as a dual barrier against HIV infection by first inactivating HIV-1 virions away from cells and then blocking HIV-1 entry on the target cell surface, indicating its potential for development as a new class of anti-HIV drug.

Phylogenetic and biological analysis of a laboratory-generated gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV).

A xenotropic murine leukemia virus-related virus (XMRV) has been reported to be an emerging pathogen associated with prostate cancer (PC) and chronic fatigue syndrome (CFS). However, recent studies have demonstrated that XMRV is a laboratory-derived virus resulting from genetic recombination between two mouse viral genomes during serial xenograft tissue transplantation. This study describes a phylogenetic analysis that compared XMRV with the ecotropic murine leukemia viruses (E-MLV), xenotropic MLV (X-MLV), and other retroviruses, including HTLV-1 and HIV-1. We found that sequences corresponding to three XMRV structural proteins (Env, Gag, and Pol) exhibited high degrees of homology with X-MLV (>91 %) and E-MLV (67-96 %), but not HTLV-1 (13-16 %) or HIV-1 (10-15 %), indicating that XMRV was derived from X-MLV and/or E-MLV. We then compared the infectivity of XMRV and E-MLV for human and murine lymphocytes, respectively. Results showed that human PBMCs were not susceptible to XMRV infection, suggesting that XMRV exhibits host cell tropism similar to E-MLV that only infects murine PBMCs. These data suggest that it is unlikely that this laboratory-generated retrovirus could cause disease in humans.

Rapid detection of SARS-CoV-2, replicating or non-replicating, using RT-PCR.

To identify animals susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection or to determine whether SARS-CoV-2 contaminated meat is from a SARS-CoV-2-infected animal, a convenient and safe method was developed for rapid detection of SARS-CoV-2 in a replicating or non-replicating status in samples using reverse transcriptase-polymerase chain reaction (RT-PCR). This strategy can also be applied to develop assays for the detection of other viruses, either replicating or not.

A quadruple protection procedure for resuming pig production in small-scale ASFV-positive farms in China.

African swine fever (ASF) outbreak has caused serious economic losses in Asia since 2018. As ASF is a new emerging disease, many farmers hesitate to raise pigs before biosafety procedures were evaluated to be effective. To support small-scale farms in resuming pig production, a comprehensive procedure, called the quadruple protection procedure (QPP), was tested in 35 small farms which had been confirmed with African swine fever virus (ASFV). The QPP takes care of the farms' construction, environmental disinfection, regular immunization, and feed quality. Qualified daily management was supplemented as well. During a one-year survey four disinfectants and one piece of equipment were used in higher frequency. A 7- or 15-day empty period after the disinfection was suitable when it was combined with the rest of the protection measures from QPP. Totally 18,730 porkers and 3,006 sows were healthy by the end of the study with percentage of 100 and 98.8, respectively, indicating that QPP could protect pigs in small-scale farms from pathogens within China. This study developed an effective protective procedure system for small-scale farms to produce pigs under the risk of ASF outbreak.

3-hydroxyphthalic anhydride-modified chicken ovalbumin exhibits potent and broad anti-HIV-1 activity: a potential microbicide for preventing sexual transmission of HIV-1.

Heterosexual transmission is the primary route by which women acquire human immunodeficiency virus (HIV)/AIDS. Thus, development of woman-controlled topical microbicides for prevention of sexual transmission of HIV is urgently needed. Here we report that 3-hydroxyphthalic anhydride-modified chicken ovalbumin (HP-OVA) exhibits potent antiviral activity against a broad spectrum of human immunodeficiency virus type 1 (HIV-1) isolates with different genotypes and biotypes. Its antiviral activity is correlated with the percentages of the chemically modified and unmodified lysines and arginines in OVA. HP-OVA inhibits HIV-1 fusion and entry through multiple mechanisms of action, including (i) blocking gp120 binding to CD4 and (ii) interfering with gp41 six-helix bundle formation. Because of the widespread availability and established safety profile of OVA, HP-OVA has good potential to be developed as an effective, safe, and affordable microbicide for prevention of HIV sexual transmission.

A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice.

HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

Combinations of the first and next generations of human immunodeficiency virus (HIV) fusion inhibitors exhibit a highly potent synergistic effect against enfuvirtide- sensitive and -resistant HIV type 1 strains.

T20 (generic name, enfuvirtide; brand name, Fuzeon) is a first-generation human immunodeficiency virus (HIV) fusion inhibitor approved for salvage therapy of HIV-infected patients refractory to current antiretroviral drugs. However, its clinical use is limited because of rapid emergence of T20-resistant viruses in T20-treated patients. Therefore, T1249 and T1144 are being developed as the second- and third-generation HIV fusion inhibitors, respectively, with improved efficacy and drug resistance profiles. Here, we found that combinations of T20 with T1249 and/or T1144 resulted in exceptionally potent synergism (combination index, <0.01) against HIV-1-mediated membrane fusion by 2 to 3 orders of magnitude in dose reduction. Highly potent synergistic antiviral efficacy was also achieved against infection by laboratory-adapted and primary HIV-1 strains, including T20-resistant variants. The mechanism underlying the synergistic effect could be attributed to the fact that T20, T1249, and T1144 all contain different functional domains and have different primary binding sites in gp41. As such, they may work cooperatively to inhibit gp41 six-helix bundle core formation, thereby suppressing virus-cell fusion. Therefore, these findings strongly imply that, rather than replacing T20, combining it with HIV fusion inhibitors of different generations might produce synergistic activity against both T20-sensitive and -resistant HIV-1 strains, suggesting a new therapeutic strategy for the treatment of HIV-1 infection/AIDS.

Fab crystallization and preliminary X-ray analysis of NC-1, an anti-HIV-1 antibody that recognizes the six-helix bundle core of gp41.

NC-1 is a murine monoclonal antibody that specifically recognizes the six-helix bundle core of the human immunodeficiency virus type 1 (HIV-1) gp41. As such, it is a useful tool for probing gp41 conformations in HIV-1 membrane fusion. To establish the structural basis underlying the NC-1 specificity, X-ray crystallography was employed to solve its three-dimensional structure. To accomplish this, hybridoma-produced NC-1 antibody was first purified and digested with papain. Its Fab fragment was then purified using size-exclusion chromatography following Fc depletion using a Protein A affinity column. Finally, crystallization of NC-1 Fab was performed by the hanging-drop vapour-diffusion method and the protein was crystallized at pH 8.0 using PEG 6000 as precipitant. The results showed that the NC-1 Fab crystals belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 118.7, c = 106.0 A. There is one Fab molecule in the asymmetric unit, with 67.5% solvent content. An X-ray diffraction data set was collected at 3.2 A resolution and a clear molecular-replacement solution was obtained for solution of the structure.

HIV-1 gp41 fusion intermediate: a target for HIV therapeutics.

Human immunodeficiency virus (HIV)-1 infection is initiated by the binding of gp120 envelope glyco-protein to its cell receptor (CD4) and a coreceptor (CXCR4 or CCR5), followed by a series of conformational changes in the gp41 transmembrane subunit. These changes include insertion of fusion peptide into the target cell membrane and association of C-heptad repeat (CHR) peptide with the N-heptad repeat (NHR) trimer, a pre-hairpin fusion intermediate. A stable six-helix bundle core is then formed, bringing the viral envelope and target cell membrane into close proximity for fusion. Peptides derived from the CHR region, such as T20 and C34, inhibit HIV-1 fusion by interacting with the gp41 fusion intermediate. A number of anti-HIV-1 peptides and small molecule compounds targeting the gp41 NHR-trimer have been identified. By combining HIV fusion/entry inhibitors targeting different sites in the gp41 fusion intermediate, a potent synergistic effect takes place, resulting in a potential new therapeutic strategy for the HIV infection/AIDS. Here, we present an overview of the current development of anti-HIV drugs, particularly those targeting the gp41 fusion intermediate.

Lessons learned from the 2019-nCoV epidemic on prevention of future infectious diseases.

Only a month after the outbreak of pneumonia caused by 2019-nCoV, more than forty-thousand people were infected. This put enormous pressure on the Chinese government, medical healthcare provider, and the general public, but also made the international community deeply nervous. On the 25th day after the outbreak, the Chinese government implemented strict traffic restrictions on the area where the 2019-nCoV had originated-Hubei province, whose capital city is Wuhan. Ten days later, the rate of increase of cases in Hubei showed a significant difference (p = 0.0001) compared with the total rate of increase in other provinces of China. These preliminary data suggest the effectiveness of a traffic restriction policy for this pandemic thus far. At the same time, solid financial support and improved research ability, along with network communication technology, also greatly facilitated the application of epidemic prevention measures. These measures were motivated by the need to provide effective treatment of patients, and involved consultation with three major groups in policy formulation-public health experts, the government, and the general public. It was also aided by media and information technology, as well as international cooperation. This experience will provide China and other countries with valuable lessons for quickly coordinating and coping with future public health emergencies.

Measures for diagnosing and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in Wuhan, China.

On 10 January 2020, a new coronavirus causing a pneumonia outbreak in Wuhan City in central China was denoted as 2019-nCoV by the World Health Organization (WHO). As of 24 January 2020, there were 887 confirmed cases of 2019-nCoV infection, including 26 deaths, reported in China and other countries. Therefore, combating this new virus and stopping the epidemic is a matter of urgency. Here, we focus on advances in research and development of fast diagnosis methods, as well as potential prophylactics and therapeutics to prevent or treat 2019-nCoV infection.