Laser spectroscopic technique for direct identification of a single virus I: FASTER CARS.

From the famous 1918 H1N1 influenza to the present COVID-19 pandemic, the need for improved viral detection techniques is all too apparent. The aim of the present paper is to show that identification of individual virus particles in clinical sample materials quickly and reliably is near at hand. First of all, our team has developed techniques for identification of virions based on a modular atomic force microscopy (AFM). Furthermore, femtosecond adaptive spectroscopic techniques with enhanced resolution via coherent anti-Stokes Raman scattering (FASTER CARS) using tip-enhanced techniques markedly improves the sensitivity [M. O. Scully, et al, Proc. Natl. Acad. Sci. U.S.A. 99, 10994-11001 (2002)].

SARS-CoV-2 causes severe epithelial inflammation and barrier dysfunction.

Infections with SARS-CoV-2 can be asymptomatic, but they can also be accompanied by a variety of symptoms that result in mild to severe coronavirus disease-19 (COVID-19) and are sometimes associated with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including interferon expression. By contrast, the adjacent endothelial layer was neither infected nor did it show productive virus replication or interferon release. With prolonged infection, both cell types were damaged, and the barrier function was deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokines or components induced during infection, which further results in the damage of the epithelial/endothelial barrier function and viral dissemination.IMPORTANCESARS-CoV-2 challenges healthcare systems and societies worldwide in unprecedented ways. Although numerous new studies have been conducted, research to better understand the molecular pathogen-host interactions are urgently needed. For this, experimental models have to be developed and adapted. In the present study we used mono cell-culture systems and we established a complex chip model, where epithelial and endothelial cells are cultured in close proximity. We demonstrate that epithelial cells can be infected with SARS-CoV-2, while the endothelium did not show any infection signs. Since SARS-CoV-2 is able to establish viremia, the link to thromboembolic events in severe COVID-19 courses is evident. However, whether the endothelial layer is damaged by the viral pathogens or whether other endothelial-independent homeostatic factors are induced by the virus is essential for understanding the disease development. Therefore, our study is important as it demonstrates that the endothelial layer could not be infected by SARS-CoV-2 in our in vitro experiments, but we were able to show the destruction of the epithelial-endothelial barrier in our chip model. From our experiments we can assume that virus-induced host factors disturbed the epithelial-endothelial barrier function and thereby promote viral spread.

Hepeliviruses in two waterbodies in Berlin, Germany.

The order Hepelivirales comprises RNA viruses of four families (Alphatetraviridae, Benyviridae, Hepeviridae, and Matonaviridae). Sequencing of virus genomes from water samples from the Havel River and the Teltow Canal (Teltowkanal) in Berlin, Germany, revealed 25 almost complete and 68 partial genomes of viruses presumably belonging to the order Hepelivirales. Only one of these viruses exhibited a relationship to a known member of this order. The members of one virus clade have a polymerase with a permuted order of the conserved palm subdomain motifs resembling the polymerases of permutotetraviruses and birnaviruses. Overall, our study further demonstrates the diversity of hepeliviruses and indicates the enzootic prevalence of hepeliviruses in unknown hosts.

A novel dicistrovirus in a captive red squirrel (Sciurus vulgaris).

Dicistroviruses are single-stranded RNA viruses in the family Dicistroviridae. The viruses have mainly been detected in arthropods and are the cause of several devastating diseases in many of these species such as honeybees. Increasingly, dicistroviruses have also been detected in both mammalian and avian species in faeces, blood and liver, but with unconfirmed pathology. Here, we report a novel dicistrovirus detected in the intestinal content of a captive red squirrel with enteritis along with the disease history, pathology and genomic characterisation of the virus. Virus particle morphology resembled those of picornaviruses with a diameter of 28-32 nm but failed to be detected using a mammalian/avian pan viral microarray. Next-generation sequencing confirmed a dicistrovirus having a typical dicistrovirus genome organization, but with the polyprotein 1 being shorter by about 100 amino acids, compared to that of other dicistroviruses. Phylogenetic analysis of ORF1 and ORF2 sequences clustered the virus with two yet unassigned dicistroviruses detected in Gorilla gorilla and a freshwater arthropod and likely to be designated to a new genus. Our data further highlights the ever-growing diversity of dicistroviruses, but the clinical significance of the virus in mammalian species and particularly red squirrels has yet to be established.

A proposed division of the family Picornaviridae into subfamilies based on phylogenetic relationships and functional genomic organization.

The highly diverse virus family Picornaviridae presently comprises 68 approved genera with 158 species plus many unassigned viruses. In order to better match picornavirus taxonomy to the functional and genomic groupings between genera, the establishment of five subfamilies (Caphthovirinae, Kodimesavirinae, Ensavirinae, Paavivirinae and Heptrevirinae) is proposed. The subfamilies are defined by phylogenetic analyses of 3CD (precursor of virus-encoded proteinase and polymerase) and P1 (capsid protein precursor) coding sequences and comprise between 7 and 22 currently approved virus genera. Due to the high within-subfamily and between-subfamily divergences of the picornavirus genera, p-distance estimates are unsuited for the demarcation of subfamilies. Members of the proposed subfamilies typically show some commonalities in their genome organisations, including VP1/2A cleavage mechanisms and possession of leader proteins. Other features, such as internal ribosomal entry site types, are more variable within and between members of genera. Some subfamilies are characterised by homology of proteins 1A, 2A, 2B and 3A encoded by members, which do not belong to the canon of orthologous picornavirus proteins. The proposed addition of a subfamily layer to the taxonomy of picornaviruses provides a valuable additional organisational level to the family that acknowledges the existence of higher-level evolutionary groupings of its component genera.

Using a mouse-adapted A/HK/01/68 influenza virus to analyse the impact of NS1 evolution in codons 196 and 231 on viral replication and virulence.

Seasonal influenza viruses circulating between 1918 and 2009 harboured two prevalent genetic variations in the NS1 coding region. A glutamic acid (E)-to-lysine (K) exchange at position 196 was reported to diminish the capacity of NS1 to control interferon induction. Furthermore, alterations at position 231 determine a carboxy-terminal extension of seven amino acids from 230 to 237 residues. Sequence analyses of NS1 of the last 90 years suggest that variations at these two positions are functionally linked. To determine the impact of the two positions on viral replication in vivo, we used a mouse-adapted variant of A/Hong Kong/01/68 (maHK68) (H3N2). maHK68 encodes an NS1 of 237 amino acids with lysine at position 196. A panel of recombinant maHK68 viruses was generated encoding NS1 variants that differed at positions 196 and 231. Our analyses showed a clear effect of the K-196-to-E exchange on interferon induction and virus virulence. These effects were further modulated by the loss of the seven-amino-acid extension. We propose that the combination of NS1 E-196 with the short C-terminal variant conferred a fitness advantage that is reflected by increased virulence in vivo. Notably, this particular NS1 constellation was observed for the pandemic 1918 H1N1 virus.

Novel enteric viruses in fatal enteritis of grey squirrels.

Astro- and kobuviruses infect both humans and animals. Here, we report on the disease history, detection and genomic characterization of novel astro- and kobuviruses from fatal diarrhoea of two juvenile grey squirrels. The virus particles had enterovirus-like morphology and a diameter of 28-32 nm. Next-generation sequencing confirmed astro- and kobuviruses and sequence analysis revealed typical astrovirus and picornavirus genome organizations. The astrovirus ORF2 sequence clustered with a clade of unassigned astroviruses, with marmot and rodent mamastroviruses as closest relatives. For the kobuvirus, divergences greater than 49.4 % for P1 and 43.5 % in the non-structural proteins indicated a novel species. However, phylogenetic analysis of the 3D polymerase showed that it clustered with that of the newly classified ludopivirus A1, suggesting a previous recombination event in the evolution of the kobuvirus. Our data provide further insights into the diversity of astro- and kobuviruses and broaden the spectrum of viruses infecting grey squirrels.

Establishment of a Highly Sensitive Assay for Detection of Hepatitis E Virus-Specific Immunoglobulins.

Hepatitis E, a liver disease caused by infection with the hepatitis E virus (HEV), is a worldwide emerging disease. The diagnosis is based on the detection of viral RNA and of HEV-specific immunoglobulins (Ig). For the latter, various assays are commercially available but still lack harmonization. In this study, a Luminex-based multiplex serological assay was established that measures the presence of total IgG, IgA, and IgM antibodies, targeting a short peptide derived from the viral E2 protein. For the validation, 160 serum samples with a known HEV serostatus were used to determine the assay cutoff and accuracy. Thereby, HEV IgG- and RNA-positive sera were identified with a sensitivity of 100% and a specificity of 98% (95% confidence interval [CI], 94% to 100%). Application of the assay by retesting 514 serum samples previously characterized with different HEV-IgG or total antibody tests revealed a high level of agreement between the assays (Cohen's kappa, 0.58 to 0.99). The established method is highly sensitive and specific and can be easily implemented in a multiplex format to facilitate rapid differential diagnostics with a few microliters of sample input.

Eurasian Avian-Like Swine Influenza A Viruses Escape Human MxA Restriction through Distinct Mutations in Their Nucleoprotein

To cross the human species barrier, influenza A viruses (IAV) of avian origin have to overcome the interferon-induced host restriction factor MxA by acquiring distinct mutations in their nucleoprotein (NP). We recently demonstrated that North American classical swine IAV are able to partially escape MxA restriction. Here we investigated whether the Eurasian avian-like swine IAV lineage currently circulating in European swine would likewise evade restriction by human MxA. We found that the NP of the influenza virus isolate A/Swine/Belzig/2/2001 (Belzig-NP) exhibits increased MxA escape, similar in extent to that with human IAV NPs. Mutational analysis revealed that the MxA escape mutations in Belzig-NP differ from the known MxA resistance cluster of the North American classical swine lineage and human-derived IAV NPs. A mouse-adapted avian IAV of the H7N7 subtype encoding Belzig-NP showed significantly greater viral growth in both MxA-expressing cells and MxA-transgenic mice than control viruses lacking the MxA escape mutations. Similarly, the growth of the recombinant Belzig virus was only marginally affected in MxA-expressing cells and MxA-transgenic mice, in contrast to that of Belzig mutant viruses lacking MxA escape mutations in the NP. Phylogenetic analysis of the Eurasian avian-like swine IAV revealed that the NP amino acids required for MxA escape were acquired successively and were maintained after their introduction. Our results suggest that the circulation of IAV in the swine population can result in the selection of NP variants with a high degree of MxA resistance, thereby increasing the zoonotic potential of these viruses. IMPORTANCE The human MxA protein efficiently blocks the replication of IAV from nonhuman species. In rare cases, however, these IAV overcome the species barrier and become pandemic. All known pandemic viruses have acquired and maintained MxA escape mutations in the viral NP and thus are not efficiently controlled by MxA. Intriguingly, partial MxA resistance can also be acquired in other hosts that express antivirally active Mx proteins, such as swine. To perform a risk assessment of IAV circulating in the European swine population, we analyzed the degree of MxA resistance of Eurasian avian-like swine IAV. Our data demonstrate that these viruses carry formerly undescribed Mx resistance mutations in the NP that mediate efficient escape from human MxA. We conclude that Eurasian avian-like swine IAV possess substantial zoonotic potential.

Viruses and atypical bacteria in the respiratory tract of immunocompromised and immunocompetent patients with airway infection.

Respiratory tract infections (RTI) can take a serious course under immunosuppression. Data on the impact of the underlying pathogens are still controversial. Samples from the upper (n = 322) and lower RT (n = 169) were collected from 136 children and 355 adults; 225 among them have been immunocompromised patients. Exclusion criteria were presence of relevant cultivable microorganisms, C-reactive protein > 20 mg/dl, or procalcitonin > 2.0 ng/ml. Samples were tested by PCR for the presence of herpesviruses (HSV-1/-2; VZV; CMV; HHV6; EBV), adenoviruses, bocaviruses, entero-/rhinoviruses (HRV), parechoviruses, coronaviruses, influenza viruses (IV), parainfluenza viruses as well as for pneumoviruses (HMPV and RSV), and atypical bacteria (Mycoplasma pneumoniae, M.p.; Chlamydia pneumoniae, C.p.). Viral/bacterial genome equivalents were detected in more than two-thirds of specimens. Under immunosuppression, herpesviruses (EBV 30.9%/14.6%, p < 0.001; CMV 19.6%/7.9%, p < 0.001; HSV-1: 14.2%/7.1%, p = 0.012) were frequently observed, mainly through their reactivation in adults. Immunocompromised adults tended to present a higher RSV prevalence (6.4%/2.4%, p = 0.078). Immunocompetent patients were more frequently tested positive for IV (15.0%/5.8%, p = 0.001) and M.p. (6.4%/0.4%, p < 0.001), probably biased due to the influenza pandemic of 2009 and an M.p. epidemic in 2011. About 41.8% of samples were positive for a single pathogen, and among them EBV (19.9%) was most prevalent followed by HRV (18.2%) and IV (16.6%). HSV-2 and C.p. were not found. Marked seasonal effects were observed for HRV, IV, and RSV. Differences in pathogen prevalence were demonstrated between immunocompetent and immunocompromised patients. The exact contribution of some herpesviruses to the development of RTI remains unclear.

Displacement of the Gent/1999 human-like swine H1N2 influenza A virus lineage by novel H1N2 reassortants in Germany.

A swine influenza survey was conducted between 2003 and 2015 in Germany. During this period, 8122 snout swabs or other respiratory specimens from pigs of 5178 herds, mainly from Germany, were investigated for the presence of swine influenza A virus (S-IAV). In total, 1310 S-IAV isolates were collected. Of this collection, the complete genome of 267 H1N2 S-IAV isolates was sequenced and phylogenetically analyzed. The data demonstrate the incursion of human-like swine H1N2 viruses (Gent/1999-like) in 2000 and prevalent circulation until 2010. From 2008 onward, a sustained and broad change of the genetic constellation of the swine H1N2 subtype commenced. The Gent/1999-like swine H1N2 viruses ceased and several new swine H1N2 reassortants emerged and became prevalent in Germany. Of these, the upsurge of the Diepholz/2008-like, Emmelsbuell/2009-like and Papenburg/2010-like viruses is notable. The data reveal the importance of reassortment events in S-IAV evolution. The strong circulation of S-IAV of different lineages in the swine population throughout the year underlines that pigs are important reservoir hosts.

Infection Studies in Pigs and Porcine Airway Epithelial Cells Reveal an Evolution of A(H1N1)pdm09 Influenza A Viruses Toward Lower Virulence.

We analyzed the virulence of pandemic H1N1 2009 influenza A viruses in vivo and in vitro. Selected viruses isolated in 2009, 2010, 2014, and 2015 were assessed using an aerosol-mediated high-dose infection model for pigs as well as air-liquid interface cultures of differentiated airway epithelial cells. Using a dyspnea score, rectal temperature, lung lesions, and viral load in the lung as parameters, the strains from 2014-2015 were significantly less virulent than the strains isolated in 2009-2010. In vitro, the viruses from 2009-2010 also differed from the 2014-2015 viruses by increased release of infectious virus, a more pronounced loss of ciliated cells, and a reduced thickness of the epithelial cell layer. Our in vivo and in vitro results reveal an evolution of A(H1N1)pdm09 viruses toward lower virulence. Our in vitro culture system can be used to predict the virulence of influenza viruses.

Macaca arctoides gammaherpesvirus 1 (strain herpesvirus Macaca arctoides): virus sequence, phylogeny and characterisation of virus-transformed macaque and rabbit cell lines.

Herpesvirus Macaca arctoides (HVMA) has the propensity to transform macaque lymphocytes to lymphoblastoid cells (MAL-1). Inoculation of rabbits with cell-free virus-containing supernatant resulted in the development of malignant lymphomas and allowed isolation of immortalised HVMA-transformed rabbit lymphocytes (HTRL). In this study, the HVMA genome sequence (approx. 167 kbp), its organisation, and novel aspects of virus latency are presented. Ninety-one open reading frames were identified, of which 86 were non-repetitive. HVMA was identified as a Lymphocryptovirus closely related to Epstein-Barr virus, suggesting the designation as 'Macaca arctoides gammaherpesvirus 1' (MarcGHV-1). In situ lysis gel and Southern blot hybridisation experiments revealed that the MAL-1 cell line contains episomal and linear DNA, whereas episomal DNA is predominantly present in HTRL. Integration of viral DNA into macaque and rabbit host cell genomes was demonstrated by fluorescence in situ hybridisation on chromosomal preparations. Analysis of next-generation sequencing data confirmed this finding. Approximately 400 read pairs represent the overlap between macaque and MarcGHV-1 DNA. Both, MAL-1 cells and HTRL show characteristics of a polyclonal tumour with B- and T-lymphocyte markers. Based on analysis of viral gene expression and immunohistochemistry, the persistence of MarcGHV-1 in MAL-1 cells resemble the latency type III, whereas the expression pattern observed in HTRL was more comparable with latency type II. There was no evidence of the presence of STLV-1 proviral DNA in MAL-1 and HTRL. Due to the similarity to EBV-mediated cell transformation, MarcGHV-1 expands the available in vitro models by simian and rabbit cell lines.

Structure-Guided Functional Annotation of the Influenza A Virus NS1 Protein Reveals Dynamic Evolution of the p85ß-Binding Site during Circulation in Humans.

Rational characterization of virulence and host-adaptive markers in the multifunctional influenza A virus NS1 protein is hindered by a lack of comprehensive knowledge about NS1-host protein protein interfaces. Here, we surveyed the impact of amino acid variation in NS1 at its structurally defined binding site for host p85ß, a regulator of phosphoinositide 3-kinase (PI3K) signaling. Structure-guided alanine scanning of all viral residues at this interface defined 10 positions contributing to the interaction, with residues 89, 95, 98, 133, 145, and 162 being the most important. A bioinformatic study of >24,000 publicly available NS1 sequences derived from viruses infecting different hosts highlighted several prevalent amino acid variants at the p85ß interface that either enhanced (I95) or weakened (N135, T145, L161, Y161, S164) p85ß binding. Interestingly, analysis of viruses circulating in humans since the 1918 pandemic revealed the temporal acquisition of functionally relevant variants at this interface. I95 (which enhanced p85ß binding) quickly became prevalent in the 1940s and experimentally conferred a fitness advantage to a recombinant 1930s-based H1N1 virus in human lung epithelial cells. Surprisingly, H1N1 and H3N2 viruses recently acquired T145 or N135, respectively, which diminished p85ß binding but apparently not the overall fitness in the human population. Evolutionary analyses revealed covariation of the NS1-p85ß binding phenotype in humans with functional changes at multiple residues in other viral proteins, suggesting an unexplored compensatory or synergistic interplay between phenotypes in vivo Overall, our data provide a resource to understand the consequences of the NS1-p85ß binding spectrum of different influenza viruses and highlight the dynamic evolution of this property in viruses circulating in humans.IMPORTANCE In humans, influenza A viruses are responsible for causing seasonal epidemics and occasional pandemics. These viruses also circulate and evolve in other animal species, creating a reservoir from which novel viruses with distinct properties can emerge. The viral nonstructural protein NS1 is an important host range determinant and virulence factor that exhibits strain-specific interactions with several host factors, although few have been characterized extensively. In the study described here, we comprehensively surveyed the impact of natural and unnatural NS1 variations on the binding of NS1 to host p85ß, a subunit of phosphoinositide 3-kinase that regulates intracellular metabolism and contributes to virus replication and virulence. We define the p85ß-binding site on NS1 and provide a predictive resource to assess this ability of NS1 in viruses from different hosts. Strikingly, we uncover a spectrum of p85ß binding by different NS1 proteins and reveal that viruses evolving in humans have undergone dynamic changes in this NS1 function over the last century.

Picornaviridae-the ever-growing virus family.

Picornaviruses are small, nonenveloped, icosahedral RNA viruses with positive-strand polarity. Although the vast majority of picornavirus infections remain asymptomatic, many picornaviruses are important human and animal pathogens and cause diseases that affect the central nervous system, the respiratory and gastrointestinal tracts, heart, liver, pancreas, skin and eye. A stunning increase in the number of newly identified picornaviruses in the past decade has shown that picornaviruses are globally distributed and infect vertebrates of all classes. Moreover, picornaviruses exhibit a surprising diversity of both genome sequences and genome layouts, sometimes challenging the definition of taxonomic relevant criteria. At present, 35 genera comprising 80 species and more than 500 types are acknowledged. Fifteen species within five new and three existing genera have been proposed in 2017, but more than 50 picornaviruses still remain unassigned.

High genetic diversity of porcine enterovirus G in Schleswig-Holstein, Germany.

Between 2012 and 2015, 495 pooled snout swabs from fattening pigs raised in Schleswig-Holstein, Germany, were screened for the presence of enterovirus G (EV-G) RNA. Nucleic acids were tested in diverse reverse transcription polymerase chain reaction assays applying published oligonucleotide primers specific for the viral protein (VP) 1 and 2/4 encoding regions as well as for 3D polymerase. Phylogenetic analyses of VP1 revealed the presence of 12 EV-G types, three of which had highly divergent sequences suggesting putative new types. Co-circulation of EV-G types was observed in several pigsties. Thus, genetic diversity of EV-G was demonstrated in this small geographic area.

Highly diverse population of Picornaviridae and other members of the Picornavirales, in Cameroonian fruit bats.

BACKGROUND: The order Picornavirales represents a diverse group of positive-stranded RNA viruses with small non-enveloped icosahedral virions. Recently, bats have been identified as an important reservoir of several highly pathogenic human viruses. Since many members of the Picornaviridae family cause a wide range of diseases in humans and animals, this study aimed to characterize members of the order Picornavirales in fruit bat populations located in the Southwest region of Cameroon. These bat populations are frequently in close contact with humans due to hunting, selling and eating practices, which provides ample opportunity for interspecies transmissions. RESULTS: Fecal samples from 87 fruit bats (Eidolon helvum and Epomophorus gambianus), were combined into 25 pools and analyzed using viral metagenomics. In total, Picornavirales reads were found in 19 pools, and (near) complete genomes of 11 picorna-like viruses were obtained from 7 of these pools. The picorna-like viruses possessed varied genomic organizations (monocistronic or dicistronic), and arrangements of gene cassettes. Some of the viruses belonged to established families, including the Picornaviridae, whereas others clustered distantly from known viruses and most likely represent novel genera and families. Phylogenetic and nucleotide composition analyses suggested that mammals were the likely host species of bat sapelovirus, bat kunsagivirus and bat crohivirus, whereas the remaining viruses (named bat iflavirus, bat posalivirus, bat fisalivirus, bat cripavirus, bat felisavirus, bat dicibavirus and bat badiciviruses 1 and 2) were most likely diet-derived. CONCLUSION: The existence of a vast genetic variability of picorna-like viruses in fruit bats may increase the probability of spillover infections to humans especially when humans and bats have direct contact as the case in this study site. However, further screening for these viruses in humans will fully indicate their zoonotic potential.

Characterization of PTV-12, a newly described porcine teschovirus serotype: in vivo infection and cross-protection studies.

Porcine teschoviruses (PTVs) constitute 1 of the 31 genera within the Picornaviridae family, comprising at least 13 genetic types (PTV-1 to PTV-13), of which only 11 (PTV-1 to PTV-11) have been recognized as serotypes to date. Specific for swine and wild boars, most PTVs are usually non-pathogenic, but some viral variants cause severe disorders in the central nervous system (Teschen disease) or milder signs (Talfan disease), as well as reproductive, digestive and respiratory disorders and skin lesions. Previous studies revealed a high diversity of teschoviruses circulating in Spanish pig populations. Phylogenetic analysis performed with these sequences and others available in GenBank disclosed 13 clusters, 11 of which corresponded to the known PTV serotypes, and 1 of 2 additional groups is represented by isolate CC25, whose full-length genomic sequence has been obtained. This group is new to science, and was putatively named PTV-12. Here, a complete characterization of this isolate is presented, including the experimental infection of minipigs to assess tissue tropism and possible pathogenicity in vivo in the host species. In addition, using this experimental animal model, we investigated whether a pre-existing infection with this PTV-12 isolate could confer cross-protection against infection with a heterotypic PTV-1 virulent strain. Based on phylogenetic analysis and serological data, we propose CC25 as the prototype strain of a new teschovirus serotype, PTV-12.

Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung.

UNLABELLED: Pigs are natural hosts for influenza A viruses and play a critical role in influenza epidemiology. However, little is known about their influenza-evoked T-cell response. We performed a thorough analysis of both the local and systemic T-cell response in influenza virus-infected pigs, addressing kinetics and phenotype as well as multifunctionality (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and cross-reactivity. A total of 31 pigs were intratracheally infected with an H1N2 swine influenza A virus (FLUAVsw) and consecutively euthanized. Lungs, tracheobronchial lymph nodes, and blood were sampled during the first 15 days postinfection (p.i.) and at 6 weeks p.i. Ex vivo flow cytometry of lung lymphocytes revealed an increase in proliferating (Ki-67(+)) CD8(+) T cells with an early effector phenotype (perforin(+) CD27(+)) at day 6 p.i. Low frequencies of influenza virus-specific IFN-γ-producing CD4(+) and CD8(+) T cells could be detected in the lung as early as 4 days p.i. On consecutive days, influenza virus-specific CD4(+) and CD8(+) T cells produced mainly IFN-γ and/or TNF-α, reaching peak frequencies around day 9 p.i., which were up to 30-fold higher in the lung than in tracheobronchial lymph nodes or blood. At 6 weeks p.i., CD4(+) and CD8(+) memory T cells had accumulated in lung tissue. These cells showed diverse cytokine profiles and in vitro reactivity against heterologous influenza virus strains, all of which supports their potential to combat heterologous influenza virus infections in pigs. IMPORTANCE: Pigs not only are a suitable large-animal model for human influenza virus infection and vaccine development but also play a central role in the emergence of new pandemic strains. Although promising candidate universal vaccines are tested in pigs and local T cells are the major correlate of heterologous control, detailed and targeted analyses of T-cell responses at the site of infection are scarce. With the present study, we provide the first detailed characterization of magnitude, kinetics, and phenotype of specific T cells recruited to the lungs of influenza virus-infected pigs, and we could demonstrate multifunctionality, cross-reactivity, and memory formation of these cells. This, and ensuing work in the pig, will strengthen the position of this species as a large-animal model for human influenza virus infection and will immediately benefit vaccine development for improved control of influenza virus infections in pigs.