Contributions of the Avian Influenza Virus HA,NA, and M2 Surface Proteins to the Induction of Neutralizing Antibodies and Protective Immunity

Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 causes severe disease and mortality in poultry. Increased transmission of H5N1 HPAIV from birds to humans is a serious threat to public health. We evaluated the individual contributions of each of the three HPAIV surface proteins, namely, the hemagglutinin (HA), the neuraminidase (NA), and the M2 proteins, to the induction of HPAIV-neutralizing serum antibodies and protective immunity in chickens. Using reverse genetics, three recombinant Newcastle disease viruses (rNDVs) were engineered, each expressing the HA, NA, or M2 protein of H5N1 HPAIV. Chickens were immunized with NDVs expressing a single antigen (HA, NA, and M2), two antigens (HA+NA, HA+M2, and NA+M2), or three antigens (HA+NA+M2). Immunization with HA or NA induced high titers of HPAIV-neutralizing serum antibodies, with the response to HA being greater, thus identifying HA and NA as independent neutralization antigens. M2 did not induce a detectable neutralizing serum antibody response, and inclusion of M2 with HA or NA reduced the magnitude of the response. Immunization with HA alone or in combination with NA induced complete protection against HPAIV challenge. Immunization with NA alone or in combination with M2 did not prevent death following challenge, but extended the time period before death. Immunization with M2 alone had no effect on morbidity or mortality. Thus, there was no indication that M2 is immunogenic or protective. Furthermore, inclusion of NA in addition to HA in a vaccine preparation for chickens may not enhance the high level of protection provided by HA.Avian influenza (AI) is an economically important disease of poultry worldwide. Avian influenza virus (AIV) belongs to the genus Influenzavirus A under the family Orthomyxoviridae. The genome of AIV consists of eight segments of single-stranded, negative-sense RNA that codes for 11 proteins (PB2, PB1, PB1-F2, PA, HA, NP, NA, M1, M2, NS1, and NS2/NEP). The genome is surrounded by the viral envelope that has two glycoprotein spikes on its outer surface, hemagglutinin (HA) and neuraminidase (NA). The HA spikes have receptor binding and fusion functions, and NA spikes have receptor-destroying activity. The envelope also contains a third integral membrane protein, M2, which is exposed on the outer surface and functions as an ion channel, essential for uncoating. The AIV surface glycoproteins are antigenically variable and are serologically divided into 16 HA (H1 to H16) and 9 NA (N1 to N9) subtypes, whereas the nonglycosylated surface protein M2 is highly conserved (9, 43). On the basis of severity of disease in poultry, AIV strains are also classified into low-pathogenic (LP) and highly pathogenic (HP) categories. Historically, highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 have caused severe disease and mortality in poultry. Recent HPAIV subtype H5N1 infections have resulted in the culling or death of more than 500 million poultry in more than 62 countries (27). Since 1997, HPAIV strains of subtype H5N1 have been found to cause disease in humans. To date, this virus has caused 436 confirmed human infections. Of these infections, 262 (60%) were fatal. Hence, HPAIV has become a major threat to both animals and humans (45). The World Organisation of Animal Health (OIE) recommends the control of HPAIV at its poultry source to decrease the viral load in susceptible avian species, thereby decreasing the risk of transmission to humans (31). The traditional method of control of HPAI has been stamping out infected flocks, which is still used in many countries, including the United States. But, due to economic reasons, culling of infected flocks is no longer considered a practical method for the control of AI in either developed or developing countries. Vaccination has been recommended by the OIE to control AI (31). Several vaccination strategies, including inactivated and live attenuated vaccines, have been evaluated for HPAIV (28). Inactivated vaccines are not commonly used because of the high cost and the difficulty in “differentiating infected from vaccinated animals” (DIVA). Live attenuated vaccines are not used because of the concern that the vaccine viruses may, through either mutation or genetic reassortment with circulating strains, become virulent (1). To overcome these difficulties, recombinant DNA technology was used to generate vectored, subunit, or DNA vaccines. Although several of these vaccines have been shown experimentally to protect against AIV, Newcastle disease virus (NDV)-vectored vaccines have shown the most promising results and also have the advantage of being bivalent vaccines against both NDV and AIV (11, 25, 32, 42). Furthermore, NDV-vectored vaccines have also been evaluated in primates with promising results (6). Newcastle disease (ND) is an economically important disease in poultry worldwide. The causative agent (NDV) is a nonsegmented, negative-strand RNA virus belonging to the genus Avulavirus in the family Paramyxoviridae. NDV strains vary greatly in virulence. Virulent NDV strains cause a severe respiratory and neurologic disease in poultry worldwide. Naturally occurring avirulent NDV strains are routinely used to control ND in many parts of world (30).We recently evaluated recombinant NDV (rNDV) expressing the HA protein of an H5N1 HPAIV vaccine (rNDV-HA) in chickens (25). Chickens immunized with rNDV-HA produced NDV- and HPAIV H5-specific antibodies and were protected against clinical disease after challenge with virulent NDV or HPAIV. Furthermore, shedding of the challenge virus was not observed, indicating complete protection. Our results demonstrated that rNDV-HA is a suitable bivalent vaccine against NDV and AIV (25). To date, all NDV-vectored vaccine studies in chickens have used HA genes derived from various HPAIV strains (11, 25, 32, 42). However, in addition to the HA protein, the envelope of AIV contains two other proteins (NA and M2) on its outer surface. Although antibodies to NA are thought not to play any role in viral attachment and penetration of the host cell, they prevent the release of virus from infected cells (20) and increase overall resistance to AIV infection in humans (37). The NA gene is thought to evolve at a lower rate than the HA gene, indicating that NA-specific antibodies may increase the breadth of protection of the HA-specific antibodies (19). The other surface protein, M2, functions as an ion channel protein and also as a target for anti-HPAIV drugs. The role of M2 protein in the induction of HPAIV-neutralizing antibodies and protective immunity is not well understood. Antibodies induced by the M2e peptide corresponding to the N-terminal 24-amino-acid ectodomain (the portion present on the virus surface) displayed broad protection against influenza A viruses of both homologous (H1N1) and heterologous (H3N1) strains in vitro and in vivo (7). However, the role of entire length of the M2 protein of AIV in induction of neutralizing antibodies and protective immunity against highly pathogenic H5N1 influenza virus in chickens has not been directly evaluated. The M2 protein is conserved among all influenza A viruses and is therefore considered an attractive target for a “universal” vaccine (8). Antibodies to HA protein alone can protect against lethal AIV challenges; the inclusion of other surface proteins in the vaccine regimen may improve the protective efficacy.In the present study, we examined the relative contribution of each of the three HPAIV surface proteins (HA, NA, and M2) to induction of neutralizing antibodies and protective immunity in chickens. Recombinant NDV vectors were constructed that individually expressed each of the three HPAIV surface proteins. They were used to immunize chickens either individually or in different possible combinations. Evaluation of the relative neutralization titers of serum antibody, shedding of challenge virus, and protection against lethal HPAIV challenge conferred by each of the NDV-vectored HPAIV surface proteins showed that HA glycoprotein was the major contributor to induction of neutralizing antibodies and protective immunity, followed by NA protein, which conferred partial protection. The M2 protein neither induced a detectable level of serum-neutralizing antibodies nor protected chickens from the HPAIV lethal challenge.     

Cytodiagnosis of erythema nodosum leprosum. A case report

BACKGROUND: The application of cytology in leprosy has been restricted to the evaluation of morphologic and bacterial indices by slit skin smears to facilitate diagnosis of cases according to the Ridley-Jopling scale. Isolated reports have now documented the use of fine needle aspiration cytology (FNAC) in the diagnosis of leprosy. CASE: A 45-year-old male presented with the abrupt onset of multiple nodular eruptions all over the body. The clinical diagnosis was Sweet's syndrome. FNAC showed numerous neutrophils in a background of foamy macrophages. Special stains revealed the presence of a large number of fragmented acid-fast bacilli in the smears. A diagnosis of erythema nodosum leprosum (ENL) was made on FNAC. CONCLUSION: The presence of neutrophils in a characteristic milieu of foamy macrophages is seen in lesions of ENL. Such a picture should prompt the cytologist to use a modified Ziehl-Neelsen stain to demonstrate acid-fast bacilli, as ENL can present as an acute episode in patients without a previous diagnosis of leprosy.     

Analysis of mixed infection of sheep with bluetongue virus serotypes 10 and 17: evidence for genetic reassortment in the vertebrate host.

Two seronegative sheep were infected intravenously with 10(9) PFU each of bluetongue virus (BTV) serotype 10 and BTV serotype 17. One animal experienced a mild bluetongue-like disease, and both experienced a short-duration viremia and developed neutralizing immune responses to both virus serotypes. Progeny virus was isolated from venous blood from each animal by using conditions in which reassortment could not have occurred during isolation. Electropherotypes were determined for the progeny viruses from the infected sheep, yielding strikingly similar results for the two animals. In both sheep, serotype 10 dominated among the progeny, accounting for 92% of the progeny. Serotype 17 was rarely isolated and accounted for 3% of the progeny analyzed. The remaining 5% of the progeny clones were reassortant and derived genome segments from both serotypes 10 and 17. Analysis of the parental origin of genome segments in the small number of reassortant progeny analyzed suggested that selection of specific genome segments may have occurred in the infected sheep. These data indicate that reassortment of genome segments occurs, at low frequency, in sheep mixedly infected with BTV.     

Kinetic estimation of the base sequence complexity of poly(A)-containing mRNA in Ehrlich-ascites-tumor cells and its abundance in nuclear RNA.

Poly(A)-containing messenger RNA was isolated from polysomes of Ehrlich ascites tumor cells, and analyzed for sequence complexity by hybridization to its complementary DNA. The results indicate the presence of about 27,000 diverse mRNA species in mouse Ehrlich ascites tumor cells. Total nuclear RNA was also hybridized to cDNA transcribed from polysomal poly(A)-containing mRNA up to an rot of 3,000 M . s. It was found that all classes of the polysomal poly(A)-containing mRNA sequences were also present in the nucleus, although the distribution varied. About 2% of the total nuclear RNA sequences were expressed as total polysomal poly(A)-containing mRNA. We also report that the total percentage of the haploid mouse genome transcribed in Ehrlich cells is significantly higher than that found in other mouse cells previously examined for poly(A)-containing mRNA sequence complexity.     

Biophysical and Biochemical Characterization of the Receptor Binding Domain of SARS-CoV-2 Variants

The Protein Journal - The newly emerging SARS-CoV-2 variants are potential threat and posing new challenges for medical intervention due to high transmissibility and escaping neutralizing antibody...