The hemagglutinin (HA) of influenza A virus plays an essential role in mediating the entry of the virus into host cells. Here, recombinant full-length HA5 protein from a H5N1 isolate (A/ chicken/hatay/2004(H5N1)) was expressed and purified from the baculovirus-insect cell system. As expected, full-length HA5 elicits strong neutralizing antibodies, as evaluated in microneutralization tests using HA5 pseudotyped lentiviral particles. In addition, two fragments of HA5 were expressed in bacteria and the N-terminal fragment, covering the ectodomain before the HA1/HA2 polybasic cleavage site, was found to elicit neutralizing antibodies. But the C-terminal fragment, which covers the remaining portion of the ectodomain, did not. Neutralizing titer of the anti-serum against the N-terminal fragment is only four times lower than the antiserum against the full-length HA5 protein. Using a novel membrane fusion assay, the abilities of these antibodies to block membrane fusion were found to correlate well with the neutralization activities.

The membrane fusion process mediated by severe acute respiratory syndrome coronavirus (SARS-CoV) S protein and its cellular receptor angiotensin-converting enzyme 2 (ACE2) had been reconstituted using two Chinese hamster ovary (CHO) cell lines that constitutively express these recombinant proteins separately. This system was applied to develop a quantitative measurement of cell-cell fusion using hepatitis C virus (HCV) NS3/4A protease and a secretion-blocked EGFP-4A/4B-SEAP (EGFP: enhanced green fluorescent protein; 4A/4B: a decapeptide substrate of NS3/4A protease; SEAP: secreted alkaline phosphatase) fusion gene. Both genes were transiently expressed in either of the CHO cell lines, followed by fusion treatment. Significant SEAP activity could be detected in the culture medium only after cell-cell fusion occurred. Cell-cell fusion provides an environment in which the protease encounters its substrate 4A/4B, thereby releasing SEAP from the fusion protein. In this study, we developed a simple, sensitive, and quantitative assay to study the membrane fusion process by measuring the extracellular activity of SEAP.

C2C12 myoblasts serve as well-established model system to study myogenesis, as they fuse to form multinucleated myotubes. Severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays a crucial role in viral entry. Exogenous expression of S protein in C2C12 myoblasts inhibits the formation of myotubes. Global changes in gene expression were studied in C2C12 cells expressing S protein using oligonucleotide microarray analysis. The expression profile showed that, most of the myogenic marker genes were downregulated. Next, we used RT-PCR analysis to reexamine some downregulated and upregulated genes. To further study the antimyogenic effects induced by the S protein, we introduced antisense Plf (proliferin), an upregulated gene, into the antimyogenic cells. Antisense Ace2 (angiotensin-converting enzyme 2), the cellular receptor of S protein, was also introduced into C2C12 myoblasts. Results indicated that antimyogenic effect induced by S protein was partially rescued in cells expressing antisense Plf, while C2C12 cells expressing antisense Ace2 showed upregulation of Plf.

The hemagglutinin (HA) of influenza A virus plays an essential role in mediating the entry of the virus into host cells. In this study, 4 HA monoclonal antibodies (MAbs) of immunoglobulin M (IgM) isotype were generated by using recombinant full-length HA protein, which was expressed and purified from the baculovirus-insect cell system, from a H5N1 isolate (A/chicken/hatay/2004(H5N1)). Western blot analysis showed that these IgM MAbs bind the HA1 subunit and prevent HA-induced agglutination of erythrocytes. Consistently, the IgM MAbs inhibits the entry of HA pseudotyped lentiviral particles into Madin-Darby Canine Kidney (MDCK) cells. The most potent MAb, MAb 4F3, was further shown to efficiently neutralize multiple clades of H5N1 influenza A virus. To our knowledge, there are few studies documenting the properties of H5N1 neutralizing antibodies of IgM isotype. Thus, this panel of MAb adds diversity to the repertoire of broadly neutralizing monoclonal antibodies that are useful for developing novel therapeutics for combating future outbreaks of H5N1.

Coronavirus disease 2019 (COVID-19), is a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the increasing number of individuals infected with COVID-19, there is a growing need for easy, dependable, accurate, scalable, and cost-effective tools. These tools should serve the purpose of population screening/surveillance, diagnosis, and prognosis. Unlike the other pandemics in the past, the current viral infection presents itself with long incubation period up to 14days. This situation is challenging because it results in an extremely high proportion of asymptomatic individuals (up to 80% and more). These individuals in turn contribute to high risk of transmission among the vulnerable population with coexisting diseases and the aged. Currently, the real time PCR test and serology is being largely employed only in symptomatic individuals. Therefore, there is a need for a dependable additional test to identify the asymptomatic individuals, to qualify them for the conrmatory rtPCR and/or serology tests. To meet these the above requirement, we decided to repurpose and test our proprietary non-invasive smartphone-based health screening mobile application Lyfas. The validated parameters captured from Lyfas were found to be inherent indicators of several cardiorespiratory, cardiovascular, autonomic nervous system, hematology and biochemistry anomalies. Apparently, these anomalies were also found and reported as early indicators of COVID-19 infection. Therefore, by means of rationale selection of these parameters, we derived a unique LYFAS_COVID_SCORE to enable detection and prioritize conrmatory testing for asymptomatic individuals. A controlled clinical study was conducted in 25(n=25) subjects to prove the hypothesis and establish the difference in LYFAS_COVID_SCORE between an infected and non-infected group of individuals. The LYFAS_COVID_SCORE derived out of this clinical study was found to be different between the two groups. The positive infected test group had a signicantly high(p=.00012) median score of 19.8(range 9.9 to 22.1) compared to 6.9(range 2.2 to 7.8) that of the healthy uninfected control group. The accuracy, sensitivity and the specicity of the study was found to be 92%, 93.2% and 90% respectively. The result of this study is therefore an evidence to use Lyfas as a potential tool in the identication, screening, and surveillance of asymptomatic individuals. The ndings from the study deserves to be extended to a large population conrmatory clinical study to add proof and value to the tool.