type-A Virus Research Articles

Broad-spectrum virucidal activity of a hydroalcoholic extract of grape pomace

Grape pomace is a by-product of winemaking often disposed of before full exploitation. Indeed, this natural product holds great potential for sustainable utilization. In this study, dried grape pomace was reprocessed to assess its potential as a valuable source of antiviral metabolites. The antiviral activity of the extract was assessed against Herpes Simplex virus type-2, Feline Calicivirus, Coxsackievirus B5, and Influenza type-A virus, revealing potent broad virucidal effect. Indeed, it abolished viral infection when the extract was incubated with viruses before or during infection, whereas no significant toxic effects were observed up to 3 mg/mL.UHPLC-MS/MS analysis identified quercetin, gallic acid, and ethyl gallate as its primary constituents. These did not fully account for its virucidal action, suggesting the presence of additional active components and/or synergistic effect of more compounds.Thus, grape pomace may be exploited as a sustainable source for the preparation of environmentally friendly broad-spectrum virucidal disinfectants.

Phylogenomic analysis uncovers a 9-year variation of Uganda influenza type-A strains from the WHO-recommended vaccines and other Africa strains

Genetic characterisation of circulating influenza viruses directs annual vaccine strain selection and mitigation of infection spread. We used next-generation sequencing to locally generate whole genomes from 116 A(H1N1)pdm09 and 118 A(H3N2) positive patient swabs collected across Uganda between 2010 and 2018. We recovered sequences from 92% (215/234) of the swabs, 90% (193/215) of which were whole genomes. The newly-generated sequences were genetically and phylogenetically compared to the WHO-recommended vaccines and other Africa strains sampled since 1994. Uganda strain hemagglutinin (n = 206), neuraminidase (n = 207), and matrix protein (MP, n = 213) sequences had 95.23–99.65%, 95.31–99.79%, and 95.46–100% amino acid similarity to the 2010–2020 season vaccines, respectively, with several mutated hemagglutinin antigenic, receptor binding, and N-linked glycosylation sites. Uganda influenza type-A virus strains sequenced before 2016 clustered uniquely while later strains mixed with other Africa and global strains. We are the first to report novel A(H1N1)pdm09 subclades 6B.1A.3, 6B.1A.5(a,b), and 6B.1A.6 (± T120A) that circulated in Eastern, Western, and Southern Africa in 2017–2019. Africa forms part of the global influenza ecology with high viral genetic diversity, progressive antigenic drift, and local transmissions. For a continent with inadequate health resources and where social distancing is unsustainable, vaccination is the best option. Hence, African stakeholders should prioritise routine genome sequencing and analysis to direct vaccine selection and virus control.

Molecular Epidemiology and Evolutionary Dynamics of Human Influenza Type-A Viruses in Africa: A Systematic Review.

Genomic characterization of circulating influenza type-A viruses (IAVs) directs the selection of appropriate vaccine formulations and early detection of potentially pandemic virus strains. However, longitudinal data on the genomic evolution and transmission of IAVs in Africa are scarce, limiting Africa’s benefits from potential influenza control strategies. We searched seven databases: African Journals Online, Embase, Global Health, Google Scholar, PubMed, Scopus, and Web of Science according to the PRISMA guidelines for studies that sequenced and/or genomically characterized Africa IAVs. Our review highlights the emergence and diversification of IAVs in Africa since 1993. Circulating strains continuously acquired new amino acid substitutions at the major antigenic and potential N-linked glycosylation sites in their hemagglutinin proteins, which dramatically affected vaccine protectiveness. Africa IAVs phylogenetically mixed with global strains forming strong temporal and geographical evolution structures. Phylogeographic analyses confirmed that viral migration into Africa from abroad, especially South Asia, Europe, and North America, and extensive local viral mixing sustained the genomic diversity, antigenic drift, and persistence of IAVs in Africa. However, the role of reassortment and zoonosis remains unknown. Interestingly, we observed substitutions and clades and persistent viral lineages unique to Africa. Therefore, Africa’s contribution to the global influenza ecology may be understated. Our results were geographically biased, with data from 63% (34/54) of African countries. Thus, there is a need to expand influenza surveillance across Africa and prioritize routine whole-genome sequencing and genomic analysis to detect new strains early for effective viral control.

Whole-genome analysis to determine the rate and patterns of intra-subtype reassortment among influenza type-A viruses in Africa.

Influenza type-A viruses (IAVs) present a global burden of human respiratory infections and mortality. Genome reassortment is an important mechanism through which epidemiologically novel influenza viruses emerge and a core step in the safe reassortment-incompetent live-attenuated influenza vaccine development. Currently, there are no data on the rate, spatial and temporal distribution, and role of reassortment in the evolution and diversification of IAVs circulating in Africa. We aimed to detect intra-subtype reassortment among Africa pandemic H1N1pdm09 (2009–10), seasonal H1N1pdm09 (2011–20), and seasonal H3N2 viruses and characterize the genomic architecture and temporal and spatial distribution patterns of the resulting reassortants. Our study was nested within the Uganda National Influenza Surveillance Programme. Next-generation sequencing was used to generate whole genomes (WGs) from 234 H1N1pdm09 (n = 116) and H3N2 (n = 118) viruses sampled between 2010 and 2018 from seven districts in Uganda. We combined our newly generated WGs with 658 H1N1pdm09 and 1131 H3N2 WGs sampled between 1994 and 2020 across Africa and identified reassortants using an automated Graph Incompatibility Based Reassortment Finder software. Viral reassortment rates were estimated using a coalescent reassortant constant population model. Phylogenetic analysis was used to assess the effect of reassortment on viral genetic evolution. We observed a high frequency of intra-subtype reassortment events, 12 · 4 per cent (94/758) and 20 · 9 per cent (256/1,224), and reassortants, 13 · 3 per cent (101/758) and 38 · 6 per cent (472/1,224), among Africa H1N1pdm09 and H3N2 viruses, respectively. H1N1pdm09 reassorted at higher rates (0.1237–0.4255) than H3N2 viruses (0 · 00912–0.0355 events/lineage/year), a case unique to Uganda. Viral reassortants were sampled in 2009 through 2020, except in 2012. 78 · 2 per cent (79/101) of H1N1pdm09 reassortants acquired new non-structural, while 57 · 8 per cent (273/472) of the H3N2 reassortants had new hemagglutinin (H3) genes. Africa H3N2 viruses underwent more reassortment events involving larger reassortant sets than H1N1pdm09 viruses. Viruses with a specific reassortment architecture circulated for up to five consecutive years in specific countries and regions. The Eastern (Uganda and Kenya) and Western Africa harboured 84 · 2 per cent (85/101) and 55 · 9 per cent (264/472) of the continent’s H1N1pdm09 and H3N2 reassortants, respectively. The frequent reassortment involving multi-genes observed among Africa IAVs showed the intracontinental viral evolution and diversification possibly sustained by viral importation from outside Africa and/or local viral genomic mixing and transmission. Novel reassortant viruses emerged every year, and some persisted in different countries and regions, thereby presenting a risk of influenza outbreaks in Africa. Our findings highlight Africa as part of the global influenza ecology and the advantage of implementing routine whole-over partial genome sequencing and analyses to monitor circulating and detect emerging viruses. Furthermore, this study provides evidence and heightens our knowledge on IAV evolution, which is integral in directing vaccine strain selection and the update of master donor viruses used in recombinant vaccine development.

Clinical observations and molecular detection of Type-A influenza virus in some of the family Equidae in eastern Saudi Arabia winter-2019.

In the current study, we are investigating the viral causes of some respiratory clinical signs in some animals belongs to the family Equidae in eastern Saudi Arabia (ESA) during winter- 2019. We observed the progression of severe respiratory clinical signs among some horses, donkeys, and ponies in the ESA. Animals showed rapid respiration, fever, nasal discharges (started as serous then changed into mucopurulent with the progression of the infection per some animals). We conducted a longitudinal study to monitor the progression of this outbreak. We conducted molecular surveillance for the influenza virus Type-A using real-time PCR and regular RT-PCR. We also conducted a serosurveillance of the virus in sera of the tested animals using the commercially available enzyme-linked immunosorbent assay (ELISA). The molecular detection of the Influenza virus type-A virus from nasal swabs of the affected animals using the real-time PCR results clearly showing that 35.1% of the tested horses, donkeys, and ponies were positives. Further confirmation was achieved by reporting the seroconversion of some of the affected animals. Several attempts were conducted to isolate the circulating influenza strains using the embryonated chicken eggs were unsuccessful. This was based on the absence of any amplicons in the harvested embryonated egg fluids using some oligonucleotides for the common influenza virus genes (HA, NA, M, and N). Meanwhile, ELISA results revealed the detection of the antibodies in sera of horses and donkeys 72.9%. Seroconversion was reported in many animals several weeks after the onset of the outbreak. Taken together all these pieces of evidence, we confirm an influenza virus type-A outbreak among the tested animals during winter 2019.

Enhanced inhibition of influenza virus infection by peptide-noble-metal nanoparticle conjugates.

The influenza (“flu”) type-A virus is a major medical and veterinary health concern and causes global pandemics. The peptide “FluPep” is an established inhibitor of influenza virus infectivity in model systems. We have explored the potential for noble-metal nanoparticle conjugates of FluPep to enhance its antiviral activity and to determine their potential as a delivery platform for FluPep. FluPep ligand is FluPep extended at its N-terminus with the sequence CVVVTAAA, to allow for its incorporation into a mixed-matrix ligand shell of a peptidol and an alkanethiol ethylene glycol consisting of 70% CVVVTol and 30% HS(CH2)11(OC2H4)4OH (mol/mol). Gold and silver nanoparticles (ca. 10 nm diameter) with up to 5% (mol/mol) FluPep ligand remained as stable as the control of mixed-matrix-passivated nanoparticles in a variety of tests, including ligand exchange with dithiothreitol. The free FluPep ligand peptide was found to inhibit viral plaque formation in canine MDCK cells (IC50 = 2.1 nM), but was less potent than FluPep itself (IC50 = 140 pM). Nanoparticles functionalised with FluPep ligand showed enhanced antiviral activity compared to the free peptides. The IC50 value of the FluPep-functionalised nanoparticles decreased as the grafting density of FluPep ligand increased from 0.03% to 5% (both mol/mol), with IC50 values down to about 10% of that of the corresponding free peptide. The data demonstrate that conjugation of FluPep to gold and silver nanoparticles enhances its antiviral potency; the antimicrobial activity of silver ions may enable the design of even more potent antimicrobial inhibitors, capable of targeting both influenza and bacterial co-infections.

Influenza epidemiology in Italy two years after the 2009–2010 pandemic

Since 2000, a sentinel surveillance of influenza, INFLUNET, exists in Italy. It is coordinated by the Ministry of Health and is divided into two parts; one of these is coordinated by the National Institute of Health (NIH), the other by the Inter-University Centre for Research on Influenza and other Transmissible Infections (CIRI-IT). The influenza surveillance system performs its activity from the 42nd week of each year (mid-October) to the 17th week of the following year (late April). Only during the pandemic season (2009/2010) did surveillance continue uninterruptedly. Sentinel physicians – about 1,200 general practitioners and independent pediatricians – send in weekly reports of cases of influenza-like illness (ILI) among their patients (over 2% of the population of Italy) to these centers. In order to estimate the burden of pandemic and seasonal influenza, we examined the epidemiological data collected over the last 3 seasons (2009–2012). On the basis of the incidences of ILIs at different ages, we estimated that: 4,882,415; 5,519,917; and 4,660,601 cases occurred in Italy in 2009–2010, 2010–2011 and 2011–2012, respectively. Considering the ILIs, the most part of cases occurred in < 14 y old subjects and especially in 5–14 y old individuals, about 30% and 21% of cases respectively during 2009–2010 and 2010–2011 influenza seasons. In 2011–2012, our evaluation was of about 4.7 million of cases, and as in the previous season, the peak of cases regarded subjects < 14 y (about 29%). A/California/07/09 predominated in 2009–2010 and continued to circulate in 2010–2011. During 2010–2011 B/Brisbane/60/08 like viruses circulated and A/H3N2 influenza type was sporadically present. H3N2 (A/Perth/16/2009 and A/Victoria/361/2011) was the predominant influenza type-A virus that caused illness in the 2011–2012 season. Many strains of influenza viruses were present in the epidemiological scenario in 2009–2012. In the period 2009–2012, overall vaccination coverage was low, never exceeding 20% of the Italian population. Among the elderly, coverage rates grew from 40% in 1999 to almost 70% in 2005–2006, but subsequently decreased, in spite of the pandemic; this trend reveals a slight, though constant, decline in compliance with vaccination. Our data confirm that 2009 pandemics had had a spread particularly important in infants and schoolchildren, and this fact supports the strategy to vaccinate schoolchildren at least until 14 y of age. Furthermore, the low levels of vaccination coverage in Italy reveal the need to improve the catch-up of at-risk subjects during annual influenza vaccination campaigns, and, if possible, to extend free vaccination to at least all 50–64-y-old subjects. Virologic and epidemiological surveillance remains critical for detection of evolving influenza viruses and to monitor the health and economic burden in all age class annually.

Design, Validation, and Absolute Sensitivity of a Novel Test for the Molecular Detection of Avian Pneumovirus

This study describes attempts to increase and measure sensitivity of molecular tests to detect avian pneumovirus (APV). Polymerase chain reaction (PCR) diagnostic tests were designed for the detection of nucleic acid from an A-type APV genome. The objective was selection of PCR oligonucleotide combinations, which would provide the greatest test sensitivity and thereby enable optimal detection when used for later testing of field materials. Relative and absolute test sensitivities could be determined because of laboratory access to known quantities of purified full-length DNA copies of APV genome derived from the same A-type virus. Four new nested PCR tests were designed in the fusion (F) protein (2 tests), small hydrophobic (SH) protein (1 test), and nucleocapsid (N) protein (1 test) genes and compared with an established test in the attachment (G) protein gene. Known amounts of full-length APV genome were serially diluted 10-fold, and these dilutions were used as templates for the different tests. Sensitivities were found to differ between the tests, the most sensitive being the established G test, which proved able to detect 6,000 copies of the G gene. The G test contained predominantly pyrimidine residues at its 3' termini, and because of this, oligonucleotides for the most sensitive F test were modified to incorporate the same residue types at their 3' termini. This was found to increase sensitivity, so that after full 3' pyrimidine substitutions, the F test became able to detect 600 copies of the F gene.

The Pattern of Burkitt's Lymphoma in Malaysian Patients.

Endemic and sporadic Burkitt's lymphoma differ in site and age of presentation, and Epstein-Barr virus (EBV) association. This study aimed to describe the clinical presentation, EBV association and p53 expression of Burkitt's lymphoma in 59 Malaysian patients. Expression of Bcl-2, retinoblastoma and Ki67 proteins was also investigated. EBV was detected by EBER in situ hybridization. Expression of p53, Bcl-2, pRb and Ki67 proteins was detected by immunohistochemical staining. Polymerase chain reaction was employed for EBV subtyping and detection of translocation t (14; 18). The male to female ratio was 3.9: 1. The most common age group was children younger than 15 years (72.9%), with a mean of 15.8y. The disease is more common in ethnic Chinese. EBV association is 33.3% and all were infected with type-A virus. Expressions of p53, Bcl-2, pRb and Ki67 proteins were detected in 42/49 (85.7%), 9/54 (16.7%), 47/51 (92.2%) and 35/39 (89.7%) of the biopsies, respectively. Translocation t (14; 18) was not detected in cases expressing the Bcl-2 protein. Clinical presentation and EBV association of Burkitt's lymphoma in Malaysian patients corresponds with that of sporadic Burkitt's lymphoma. This study also reveals that a substantial proportion of cases express high levels of p53 and retinoblastoma proteins, in contrast to the Bcl-2 protein.

Inactivation Effect of Tea Leaf Catechins on Human Type-A Influenza Virus.

The effects of catechins obtained from the hot water extract of green tea leaves on two human type-A influenza virus strains of Aichi/2/68 and PR/8/34 were studied. In this study, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), the crude catechins containing these catechins and (+)-catechin were used. Consequently it was observed that catechins used in this study had an inhibitory effect on the hemolytic activity to red blood cells induced by these two type-A virus strains under acidic conditions (pH 5.1 or 5.4) although they did not have an influence on the agglutination activity to red blood cells induced by the same virus strains. After incubation of (-)-ECg and (-)-EGCg with A/Aichi/2/68 virus, MDCK cells, virus-sensitive cells, were infected with the virus and the ability of virus proliferation was measured in terms of an index of the agglutination activity of the virus to red blood cells. The used two catechins, (-)-ECg and (-)-EGCg, inhibited the virus proliferation at concentrations of 100μg/ml and 50μg/ml, respectively. Furthermore, at a concentration of 2.0 mg/ml these two catechins were found to inhibit the activity of neuraminidase on the surface of the virus membrane by 71.3% and 60.8%, respectively. From the above-mentioned results, it is considered that among the catechins contained in green tea leaves, such two catechins as (-)-ECg and (-)-EGCg inhibit the activity of neuraminidase present on the surface of human influenza virus in order to block the invasion of the influenza virus into virus-sensitive cells.

Epstein‐barr virus (EBV) in malaysian upper‐aerodigestive‐tract lymphoma: Incidence and sub‐type

Epstein-Barr virus (EBV) type B, a less potent transformer of B lymphocytes than type A, has rarely been detected in EBV-associated neoplasms except in AIDS-related lymphomas, in which about 50% of the cases contained this sub-type. In this study we analyzed the association of EBV and the distribution of virus sub-types in Asian non-Hodgkin's lymphoma (NHL) of the upper aerodigestive tract. We studied archival material of 29 NHL cases from Malaysia. B- and T-cell associated antigens were demonstrated by immunohistochemistry, and EBV early RNA EBER-1 was demonstrated using the RNA in situ hybridization technique. EBV was detected in the majority of tumour cells in 11/13 T-NHL but in only 1/16 B-NHL. EBV was sub-typed by single-step polymerase chain reaction of the EBNA-2 gene. This was successful in 9/10 cases of EBER-1-positive tumours and all contained type-A virus only. Our results showed a preponderance of T-cell lymphoma of the upper aerodigestive tract in the ethnic Chinese group of Malaysian patients, and EBV was strongly associated with T-NHL but not with B-NHL. Our results suggest that type-A EBV is the prevalent sub-type in Asian NHL of the upper aerodigestive tract, similarly to findings in Asian nasopharyngeal carcinoma.

Detection of A-Type and B-Type Epstein-Bart Virus in Throat Washings and Lymphocytes

Peripheral blood lymphocytes, obtained from 94 individuals, were assayed for the presence and type of Epstein-Barr virus (EBV) using primers specific for A-type and B-type EBV in the polymerase chain reaction (PCR). Samples from 16 individuals (17%) were negative for EBV sequences. Of the remaining individuals A-type EBV was detected in 35%, B-type in 21%, and both A- and B-type EBV in 27%. Samples of throat washings were also collected from 33 healthy donors and the presence and type of EBV was determined using PCR. EBV was not detected in 12 donors. However, of those who were excreting EBV, A-type EBV was present in 11 donors (52%), B-type in 7 donors (33%), and both A- and B-types in the remaining 3 donors (14%). These results suggest that infection with B-type EBV and coinfections with both A- and B-type EBV are more common than previously thought.

Epstein‐barr virus genotypes in NPC biopsies from North Africa

The genotypes of Epstein-Barr virus (EBV) were investigated in North African nasopharyngeal carcinoma (NPC) biopsies, nasopharyngeal chronic inflammation (NCI) biopsies, and saliva of healthy individuals from Algeria and Tunisia where there is an intermediate incidence of NPC. The prevalence of A-type virus in NPC, NCI biopsies and saliva of healthy individuals was found in these regions by means of a PCR assay. Restriction enzyme polymorphism analysis by Southern blotting revealed that all North African EBV variants have a conserved restriction site on BamHI W'-I' and XhoI LMP gene. No additional BamHI enzyme site on the BamHI-F fragment was observed; however, the presence of an extra BamHI site on the BamHI-H fragment giving 2 HI and H2 fragment-like EBV M-ABA strains was found. All EBV strains present in NPC or NCI biopsies at all ages were homogeneous in these polymorphisms and no correlation was observed between the EBV genotypes from NPC patients and clinical stages of the cancer. These characteristics revealed a significant difference between the EBV variants common in Chinese NPC and those in North African NPC.

Patterns of reactivity of Epstein-Barr virus-specific T cells in A-type donor cultures after reactivation with autologous A- or B-type transformants

Regression endpoints were assessed in cultures from 11 Epstein-Barr (EB) virus A-type sero-positive donors and 2 seronegative donors using A- and B-type EB virus preparations. In 9 11 of the seropositive donors, the resulting endpoints using A-type or B-type virus were similar and demonstrated a significant T cell response to both virus types. However, the regression endpoints for 2 11 seropositive donors were reproducibly higher with B-type virus compared with A-type virus, indicating a weak T cell response to the B-type virus compared with that to the A-type virus. Seronegative donor cultures showed no regression. The patterns of reactivity of bulk cultures of EB virus-specific cytotoxic T cells and T cell clones from selected seropositive donors were compared. Four of six donors showed evidence of a cytotoxic T cell response to A- and B-type autologous transformants while cytotoxic T cells from 2 6 donors (corresponding to those identified as lacking regression to B-type virus) lysed autologous targets infected with A-type but not B-type virus. The results show that while most A-type seropositive donors are capable of mounting a T cell response to A- and B-type virus, certain donors apparently lack B-type reactivity.

Enhancement of DTH Response by Cholera Toxin B Subunit Inoculated Intranasally Together with Influenza HA Vaccine

The effects of B subunit of cholera toxin (CTB) on delayed-type hypersensitivity (DTH) response to influenza vaccine derived from influenza virus A/PR/8/34 (PR-8, HlNl) virus were investigated in B10 mice that were immunized intranasally with both influenza vaccine and CTB. The result showed that intranasal inoculation of this combination augmented DTH response to influenza vaccine, which reached its peak 6 days after inoculation, and also induced accelerated DTH response upon a second inoculation of influenza vaccine alone 4 weeks later, that the cross-reactive DTH response to PR-8 vaccine was elicited by the injection of the different influenza A-type virus vaccine into the footpad of the vaccinated mice, but was not by influenza B-type virus vaccine, that the DTH-mediating T cells were detected selectively in the lungs of mice that received the nasal inoculation of the vaccine and CTB together, but that subcutaneous inoculation of this combination failed to induce DTH-mediating T cells in the lungs. These results, together with the previous papers (Tamura et al, Vaccine 7: 257-262; 314-320, 1989), suggest that CTB could augment both humoral and DTH responses against influenza vaccine in the respiratory mucosal tract.

Protection against influenza virus infection by a two-dose regimen of nasal vaccination using vaccines combined with cholera toxin B subunit

The effectiveness of the two-dose regimen, composed of a primary intranasal inoculation of influenza A-type virus HA vaccine together with B subunit of cholera toxin (CTB) and the subsequent intranasal inoculation of vaccine alone 4 weeks later, was examined. In mice given a relatively high dose of virus A/PR/8/34 (PR-8, H1N1) HA vaccine (1.5 μg) both as a primary antigen with CTB (1 μg) and as the second antigen, the secondary responses of both antiviral IgA antibodies in nasal wash and haemagglutination-inhibiting (HI) antibody in serum were much higher than those of primary responses and persisted for > 12 weeks after the second inoculation. Even in mice that received reduced amounts of a primary vaccine (0.03 μg) [prepared from virus PR-8, A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2) together with reduced amounts of CTB (0.05 μg), the subsequent inoculation of PR-8 vaccine produced both nasal IgA and serum HI antibodies and provided complete protection against homologous A-type virus (PR-8) infection. Moreover, the combination of the reduced amounts of heterologous A-type virus vaccine (A/Yamagata or A/Fukuoka) with CTB for primary inoculation and the secondary heterologous A-type virus vaccine [A/Yamagata, A/Kumamoto/37/79 (H1N1), or A/Fukuoka] resulted in high levels of cross-reactive IgA antibodies and partial cross-protection against PR-8 infection. On the other hand, a second inoculation of B/Ibaraki/2/85 vaccine failed to produce cross-reactive antibodies and to protect against PR-8 infection. These results suggest that a two-dose regimen using A-type virus vaccines together with a small dose of CTB can provide not only complete protection against homologous virus infection, but also partial cross-protection against heterologous A-type virus infection, in parallel with high persistent levels of cross-reactive IgA antibodies.

Induction of influenza A virus cross-reactive cytotoxic T cells by a nucleoprotein/haemagglutinin preparation.

An ammonium deoxycholate fraction from bromelain-treated influenza A virus was highly enriched for virus nucleoprotein and contained residual haemagglutinin (NP/HA). The preparation did not contain detectable levels of matrix or neuraminidase proteins and was free of infectious virus. NP/HA effectively primed mice for cytotoxic T cells which lysed syngeneic cells infected with any type A influenza virus. Furthermore, NP/HA generated A-type virus cross-reactive cytotoxic T cells when added in vitro to spleen cells from mice previously primed with infectious influenza A virus. These properties imply that NP/HA has potential as a vaccine for heterotypic influenza A immunity.

Quantitation of influenza virus antigens on infected target cells and their recognition by cross-reactive cytotoxic T cells.

Monoclonal antibody to type-A influenza virus matrix (M)-protein was used to quantitate the appearance of M-protein on abortively infected P815 cells. After 16 h of infection with different type-A viruses, only a low amount of M-protein appears on the surface of infected cells (approximately 10(3) site/cell) in contrast to approximately 10(5) hemagglutinin molecules on each cell surface. However, virus replication is required for M-protein appearance. Analysis of solubilized membranes purified from 16-h-infected cells shows approximately 10(4) M-protein molecule/cell in the plasma membrane, a content that is consistent with the observed low surface expression, and that indicates that most of the M-protein is localized internally. We found no evidence that cross-reactive cytotoxic T cells could recognize M-protein; neither monoclonal antibody or hyperimmune anti-M-protein antiserum could inhibit T cell killing, either alone or in combination with monoclonal anti-H-2 antibody. Taken together, the low level of M-protein appearance and lack of T cell blocking by anti-M-protein antibody leaves doubt that M-protein is the antigen recognized by cross-reactive cytotoxic T cells.

Epithelial cell line and subline established from premalignant mouse mammary tissue

A cell line and subline with epithelial characteristics were established from mouse mammary hyperplastic alveolar nodules (HAN). The cells do not grow in suspension cultures in vitro or form tumors in vivo. The cells do produce significant amounts of C-type and A-type virus and low amounts of plasminogen activator.

Paracrystalline structures in the oocytes of a mouse, mus cervicolor poppaeus

Large and multiple paracrystalline bodies exist in the cytoplasm of the oocytes of a wild mouse strain originating in Thailand, Mus cervicolor poppaeus. The structural details of these crystals are identical to much smaller paracrystalline structures described in preimplantation embryos of the laboratory mouse, Mus musculus (Enders and Schlafke, 1965; Calarco and Brown, 1969; Calarco and Szöllösi, 1973). These latter often occur in close proximity or even in contact with the rough endoplasmic reticulum (RER). A number of A-type virus like particles bud into these cirternae. In spite of the fact that thus far no intracisternal A-type particles were recognized in oocytes of Mus cervicolor poppaeus the similarity between the two paracrystalline structures warrants their description and comparison.In the cytoplasm of oocytes we collected by puncture from large Graafian follicles of Mus cervicolor poppaeus. In one micron sections stained with Richardson´s strain (Richardson et al., 1960), rhomboidal crystalline bodies of various sizes can be recognized. The crystalloids stain intensely blue (Fig. 1).