Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA.

The piscine orthomyxovirus called infectious salmon anemia virus (ISAV) is one of the most important emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV, which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization and study of this virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral, and mammalian genetic elements included in the pSS-URG vectors allow the expression of the eight viral RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious rISAV in salmon cells.

Novel quaranjavirus and other viral sequences identified from ticks parasitizing hunted wildlife in Trinidad and Tobago.

Hunters are at a higher risk for exposure to zoonotic pathogens due to their close interactions with wildlife and arthropod vectors. In this study, high throughput sequencing was used to explore the viromes of two tick species, Amblyomma dissimile and Haemaphysalis juxtakochi, removed from hunted wildlife in Trinidad and Tobago. We identified sequences from 3 new viral species, from the viral families Orthomyxoviridae, Chuviridae and Tetraviridae in A. dissimile.

Within-Host and Between-Host Evolution in SARS-CoV-2-New Variant's Source.

Some of the newly emerging corona viral variants show high numbers of mutations. This is unexpected for a virus with a low mutation rate due to an inherent proof-reading system. Could such a variant arise under very special conditions occurring in a host where the virus replicates and mutates in a rather unlimited fashion, such as in immune compromised patients? The virus was shown to replicate in an immunosuppressed cancer patient for more than 105 days and might be a source of new variants. These patients are asymptomatic and the virus may therefore escape detection and attention and be high-risk. Similarly, HIV-infected individuals may be immunocompromised and support coronavirus replication with increased mutation rates. The patients may promote "within-host evolution". Some of the viruses present in such a highly mutagenic swarm or quasispecies within one patient may become founders and cause a pandemic by further "between-host evolution". B.1.1.7 with 23 mutations may be such a case. Immunosuppressed patients can be identified and treated by the synthetic antibody cocktails as passive immunization and kept under control. Immunosuppressed patients can be easily identified and supervised by healthcare workers-once they become aware of the risk-to avoid new variants with pandemic potential.

Typical epidemiology of respiratory virus infections in a Brazilian slum.

Host population size, density, immune status, age structure, and contact rates are critical elements of virus epidemiology. Slum populations stand out from other settings and may present differences in the epidemiology of acute viral infections. We collected nasopharyngeal specimens from 282 children aged ≤5 years with acute respiratory tract infection (ARI) during 2005 to 2006 in one of the largest Brazilian slums. We conducted real-time reverse transcription-polymerase chain reaction (RT-PCR) for 16 respiratory viruses, nested RT-PCR-based typing of rhinoviruses (HRVs), and collected clinical symptoms. Viruses were common causes of respiratory disease; with ≥1 virus being detected in 65.2% of patients. We detected 15 different viruses during 1 year with a predominance of HRV (33.0%) and human respiratory syncytial virus (hRSV, 12.1%) infections, and a high rate of viral coinfections (28.3%). We observed seasonality of hRSV, HRV and human coronavirus infections, more severe symptoms in hRSV and influenza virus (FLU) infections and prolonged circulation of seven HRV clusters likely representing distinct serotypes according to genomic sequence distances. Potentially unusual findings included the absence of human metapneumovirus detections and lack of typical FLU seasonal patterns, which may be linked to the population size and density of the slum. Nonetheless, most epidemiological patterns were similar to other studies globally, suggesting surprising similarities of virus-associated ARI across highly diverse settings and a complex impact of population characteristics on respiratory virus epidemiology.

Viral etiology of acute respiratory infections in children in Southern Iran

Abstract INTRODUCTION: Prevalence of influenza A virus (Flu-A), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) was assessed in children with acute respiratory infections (ARIs). METHODS: Nasopharyngeal aspirates and throat swabs were subjected to real-time polymerase chain reaction (PCR) to detect RSV and Flu-A and to conventional PCR to detect hMPV. RESULTS: Of the 156 children assessed, 93 (59.6%) carried at least one virus, with 35.9% positive for RSV, 14.1% for hMPV, and 9.6% for Flu-A. The prevalence of co-infections was 2.6%. CONCLUSIONS: The high detection rate may reflect increased sensitivity of real-time PCR compared to traditional PCR and viral culture.

Sinu virus, a novel and divergent orthomyxovirus related to members of the genus Thogotovirus isolated from mosquitoes in Colombia.

The genome and structural organization of a novel insect-specific orthomyxovirus, designated Sinu virus, is described. Sinu virus (SINUV) was isolated in cultures of C6/36 cells from a pool of mosquitoes collected in northwestern Colombia. The virus has six negative-sense ssRNA segments. Genetic analysis of each segment demonstrated the presence of six distinct ORFs encoding the following genes: PB2 (Segment 1), PB1, (Segment 2), PA protein (Segment 3), envelope GP gene (Segment 4), the NP (Segment 5), and M-like gene (Segment 6). Phylogenetically, SINUV appears to be most closed related to viruses in the genus Thogotovirus.

Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia.

UNLABELLED: Tilapia are an important global food source due to their omnivorous diet, tolerance for high-density aquaculture, and relative disease resistance. Since 2009, tilapia aquaculture has been threatened by mass die-offs in farmed fish in Israel and Ecuador. Here we report evidence implicating a novel orthomyxo-like virus in these outbreaks. The tilapia lake virus (TiLV) has a 10-segment, negative-sense RNA genome. The largest segment, segment 1, contains an open reading frame with weak sequence homology to the influenza C virus PB1 subunit. The other nine segments showed no homology to other viruses but have conserved, complementary sequences at their 5' and 3' termini, consistent with the genome organization found in other orthomyxoviruses. In situ hybridization indicates TiLV replication and transcription at sites of pathology in the liver and central nervous system of tilapia with disease. IMPORTANCE: The economic impact of worldwide trade in tilapia is estimated at $7.5 billion U.S. dollars (USD) annually. The infectious agent implicated in mass tilapia die-offs in two continents poses a threat to the global tilapia industry, which not only provides inexpensive dietary protein but also is a major employer in the developing world. Here we report characterization of the causative agent as a novel orthomyxo-like virus, tilapia lake virus (TiLV). We also describe complete genomic and protein sequences that will facilitate TiLV detection and containment and enable vaccine development.

Characterization of TcCYC6 from Trypanosoma cruzi, a gene with homology to mitotic cyclins.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi.

Incidence of viral respiratory infections in a prospective cohort of outpatient and hospitalized children aged ≤5 years and its associated cost in Buenos Aires, Argentina.

BACKGROUND: Although information about the incidence of viral respiratory illnesses and their associated cost can help health officials explore the value of interventions, data are limited from middle-income countries. METHODS: During 2008-2010, we conducted a prospective cohort study and followed ~1,800 Argentinian children aged ≤5 years to identify those children who were hospitalized or who sought care at an emergency room with any acute respiratory infection sign or symptom (e.g., rhinorrhea, cough, wheezing, tachypnea, retractions, or cyanosis). Respiratory samples were obtained for respiratory syncytial virus, influenza, parainfluenza, adenovirus, and metapneumovirus testing by immunofluorescence and for rhinovirus by real-time reverse transcription polymerase chain reaction. RESULTS: The incidence of respiratory syncytial virus (24/1000 children-years), human metapneumovirus (8/1000 children-years), and influenza (8/1000 children-years) illnesses was highest among hospitalized children aged <6 months and decreased among older children. In contrast, the incidence of rhinovirus was highest (12/1000 children-years) among those aged 6-23 months. In the emergency room, the incidence of rhinovirus was 459; respiratory syncytial virus 352; influenza 185; parainfluenza 177; metapneumovirus 130; and adenovirus 73/1,000 children-years. The total cost of hospitalization was a median of US$529 (Interquartile range, US$362-789). CONCLUSIONS: Our findings indicate that respiratory viruses, in particular rhinovirus, respiratory syncytial virus, metapneumovirus, and influenza may be associated with severe illness causing substantial economic burden.

Vaccination using recombinants influenza and adenoviruses encoding amastigote surface protein-2 are highly effective on protection against Trypanosoma cruzi infection.

In the present study we evaluated the protection raised by immunization with recombinant influenza viruses carrying sequences coding for polypeptides corresponding to medial and carboxi-terminal moieties of Trypanosoma cruzi ´s amastigote surface protein 2 (ASP2). Those viruses were used in sequential immunization with recombinant adenovirus (heterologous prime-boost immunization protocol) encoding the complete sequence of ASP2 (Ad-ASP2) in two mouse strains (C57BL/6 and C3H/He). The CD8 effector response elicited by this protocol was comparable to that observed in mice immunized twice with Ad-ASP2 and more robust than that observed in mice that were immunized once with Ad-ASP2. Whereas a single immunization with Ad-ASP2 sufficed to completely protect C57BL/6 mice, a higher survival rate was observed in C3H/He mice that were primed with recombinant influenza virus and boosted with Ad-ASP2 after being challenged with T. cruzi. Analyzing the phenotype of CD8+ T cells obtained from spleen of vaccinated C3H/He mice we observed that heterologous prime-boost immunization protocol elicited more CD8+ T cells specific for the immunodominant epitope as well as a higher number of CD8+ T cells producing TNF-α and IFN-γ and a higher mobilization of surface marker CD107a. Taken together, our results suggest that immunodominant subpopulations of CD8+ T elicited after immunization could be directly related to degree of protection achieved by different immunization protocols using different viral vectors. Overall, these results demonstrated the usefulness of recombinant influenza viruses in immunization protocols against Chagas Disease.

Clinical characteristics and outcomes of influenza and other influenza-like illnesses in Mexico City.

BACKGROUND: Influenza-like illnesses (ILI) are estimated to cause millions of deaths annually. Despite this disease burden, the etiologic causes of ILI are poorly described for many geographical regions. METHODS: Beginning in April 2010, we conducted an observational cohort study at five hospitals in Mexico City, enrolling subjects who met the criteria for ILI. Evaluations were conducted at enrollment and on day 28, with the collection of clinical data and a nasopharyngeal swab (or nasal aspirate in children). Swabs were tested by multiplex PCR for 15 viral pathogens and real-time PCR for influenza. RESULTS: During the first year, 1065 subjects were enrolled in this study, 55% of whom were hospitalized; 24% of all subjects were children. One or more pathogens were detected by PCR in 64% of subjects, most commonly rhinovirus (25% of all isolates) and influenza (24% of isolates). Six percent of subjects died, and of those, 54% had no pathogen identified. Rhinovirus was the most common pathogen among those who died, although it did not have the highest case fatality rate. CONCLUSIONS: Multiple respiratory viruses beyond influenza are associated with significant morbidity and mortality among adults and children in Mexico City. Detection of these agents could be useful for the adjustment of antibiotic treatment in severe cases.

Novel strategy to evaluate infectious salmon anemia virus variants by high resolution melting.

Genetic variability is a key problem in the prevention and therapy of RNA-based virus infections. Infectious Salmon Anemia virus (ISAv) is an RNA virus which aggressively attacks salmon producing farms worldwide and in particular in Chile. Just as with most of the Orthomyxovirus, ISAv displays high variability in its genome which is reflected by a wider infection potential, thus hampering management and prevention of the disease. Although a number of widely validated detection procedures exist, in this case there is a need of a more complex approach to the characterization of virus variability. We have adapted a procedure of High Resolution Melting (HRM) as a fine-tuning technique to fully differentiate viral variants detected in Chile and projected to other infective variants reported elsewhere. Out of the eight viral coding segments, the technique was adapted using natural Chilean variants for two of them, namely segments 5 and 6, recognized as virulence-associated factors. Our work demonstrates the versatility of the technique as well as its superior resolution capacity compared with standard techniques currently in use as key diagnostic tools.

Viral etiology among the elderly presenting acute respiratory infection during the influenza season.

INTRODUCTION: Acute respiratory tract infections are the most common illness in all individuals. Rhinoviruses have been reported as the etiology of more than 50% of respiratory tract infections worldwide. The study prospectively evaluated 47 elderly individuals from a group of 384 randomly assigned for acute respiratory viral infections (cold or flu) and assessed the occurrence of human rhinovirus (HRV), influenza A and B, respiratory syncytial virus and metapneumovirus (hMPV) in Botucatu, State of São Paulo, Brazil. METHODS: Forty-nine nasal swabs collected from 47 elderly individuals following inclusion visits from 2002 to 2003 were tested by GenScan RT-PCR. HRV-positive samples were sequenced for phylogenetic analysis. RESULTS: No sample was positive for influenza A/B or RSV. HRV was detected in 28.6% (14/47) and hMPV in 2% (1/47). Of 14 positive samples, 9 isolates were successfully sequenced, showing the follow group distribution: 6 group A, 1 group B and 2 group C HRVs. CONCLUSIONS: The high incidence of HRV during the months of the influenza season requires further study regarding HRV infection impact on respiratory complications among this population. Infection caused by HRV is very frequent and may contribute to increasing the already high demand for healthcare during the influenza season.

Viral etiology among the elderly presenting acute respiratory infection during the influenza season / Etiologia viral de infecção respiratória aguda entre idosos, durante a temporada de gripe

INTRODUCTION: Acute respiratory tract infections are the most common illness in all individuals. Rhinoviruses have been reported as the etiology of more than 50 percent of respiratory tract infections worldwide. The study prospectively evaluated 47 elderly individuals from a group of 384 randomly assigned for acute respiratory viral infections (cold or flu) and assessed the occurrence of human rhinovirus (HRV), influenza A and B, respiratory syncytial virus and metapneumovirus (hMPV) in Botucatu, State of São Paulo, Brazil. METHODS: Forty-nine nasal swabs collected from 47 elderly individuals following inclusion visits from 2002 to 2003 were tested by GenScan RT-PCR. HRV-positive samples were sequenced for phylogenetic analysis. RESULTS: No sample was positive for influenza A/B or RSV. HRV was detected in 28.6 percent (14/47) and hMPV in 2 percent (1/47). Of 14 positive samples, 9 isolates were successfully sequenced, showing the follow group distribution: 6 group A, 1 group B and 2 group C HRVs. CONCLUSIONS: The high incidence of HRV during the months of the influenza season requires further study regarding HRV infection impact on respiratory complications among this population. Infection caused by HRV is very frequent and may contribute to increasing the already high demand for healthcare during the influenza season.

INTRODUÇÃO: Infecções agudas do trato respiratório estão entre as doenças mais comuns em todas as pessoas. Os rinovírus têm sido descritos como agente etiológico de mais de 50 por cento das infecções do trato respiratório ao redor do mundo. O objetivo deste trabalho foi avaliar a ocorrência de rinovírus humano (HRV), influenza vírus A e B, vírus respiratório sincicial humano e metapneumovírus (hMPV) em uma população de idosos que apresentava sintomas de gripe ou resfriado, e que residiam na Cidade de Botucatu, Estado de São Paulo, Brasil. MÉTODOS: Foram coletados swabs nasais de 47 idosos após visitas de inclusão, entre os anos de 2002 e 2003 e que foram testadas através de GeneScan RT-PCR. RESULTADOS: HRV foi detectado em 28.6 por cento (14/47) e hMPV em 2 por cento (1/47). De 14 amostras positivas para HRV, 9 foram sequenciadas, mostrando a seguinte distribuição de grupos: grupo A: 6 amostras, grupo B: 1 amostra e grupo C: 2 amostras. CONCLUSÕES: A alta incidência de HRV durante os meses de ocorrência de gripe necessita de estudos posteriores para avaliar o impacto desse vírus entre os idosos. A alta frequência de HRV pode contribuir para o aumento da demanda por serviços de saúde durante a estação de influenza.

[The origin of the genetic variability of influenza viruses]. / El origen de la variabilidad genética de los virus de la influenza.

To better understand the significant variability displayed by influenza viruses, we need to be aware not only of its genetic characteristics, but also of the effect this genetic makeup has on proteins associated with viral replication and antigenicity. The origin of such diversity is due first and foremost to its segmented genome that allows segment reassortment (antigenic shift) and second to the error prone viral polymerase (antigenic drift) responsible of copying the genes enclosed in these segments. These two combined mechanisms confer a genetic plasticity that often leads to the emergence of new influenza viruses in nature.

Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity.

In this work, we explored an original vaccination protocol using recombinant influenza and adenovirus. We constructed recombinant influenza viruses harboring dicistronic NA segments containing the surface antigen 2 (SAG2) from Toxoplasma gondii under control of the duplicated 3' promoter. Recombinant influenza viruses were able to drive the expression of the foreign SAG2 sequence in cell culture and to replicate efficiently both in cell culture and in lungs of infected mice. In addition, mice primed with recombinant influenza virus and boosted with a recombinant adenovirus encoding SAG2 elicited both humoral and cellular immune responses specific for SAG2. Moreover, when immunized animals were challenged with the cystogenic P-Br strain of T. gondii, they displayed up to 85% of reduction in parasite burden. These results demonstrate the potential use of recombinant influenza vectors harboring the dicistronic segments in the development of vaccines against infectious diseases.