Molecular characteristics of fowl adenovirus strains detected in broiler chickens on diets without immunostimulant supplements.

Introduction: Outbreaks of fowl adenovirus (FAdV) infection in chicken flocks in Poland threaten birds' health and lives and are rising in frequency. The risk of these infections in immunocompromised poultry flocks with developed clinical symptoms was analysed through virus detection in broiler chicks and correlation of cases with the birds' immune strength. Material and Methods: Samples were analysed from four broiler farms with chicks from the same hatchery in Silesia, Poland where feeding regimes were different. A normal diet was provided to birds on the control farm; a normal diet and probiotic, prebiotic, vitamin and microelement supplementation was supplied on another farm; a normal diet and antibiotics on the third; and a normal diet and both forms of supplementation were given on the fourth farm. Amplification of the virus DNA in a PCR with hexon gene L1 loop hypervariable region 1-4 primers determined the molecular characteristics of isolates of adenovirus strains obtained from necropsy tissue samples. The amplicon sequences were analysed, the pair-wise distances were determined, the maximum likelihood estimate for the gamma parameter for site rates was produced, Tajima's D neutrality test was run and the relative synonymous codon usage and transition/transversion bias were calculated. Results: Two species and two serotypes of fowl adenovirus - MW353018-FAdV-1/A-L-liver and MW353019-FAdV-5/B-I-intestine - were isolated in three-week-old broiler chicks on the control farm. Conclusion: Supplementation of broiler chicken flocks with probiotics, prebiotics, vitamins and microelements may have a significant beneficial effect on immunity and can prevent virus infection. The studies provided new information on the molecular characteristics of adenovirus strains isolated from chicks with a low level of immunity.

Probiotic supplementation as an alternative to antibiotics in broiler chickens.

Introduction: The broiler chicken digestive tract microbiome maintains the bird's immunity. Its composition has been shown to be important not only for the immune system but also for the gastrointestinal function and productivity of broiler chickens. If the microbiome is populated by supplementation with Lactobacillus, Pediococcus and Saccharomyces spp. - microorganisms with probiotic properties and alternatives to antibiotics - the immune system is stimulated. The use of probiotic supplements in the broiler production cycle can boost bird immunity and prevent adenovirus infection. The resilience of broiler chickens in different feeding schemes including supplementation with these microorganisms was assessed. Material and Methods: Four groups of Ross 308 chickens vaccinated on the standard scheme were investigated over 42 days. Group P received probiotics, prebiotics and vitamins; group AO received antibiotics; group P&AO received probiotics, prebiotics, vitamins and antibiotics; and the control group C received none of these. The birds' immunocompetence against common viral poultry pathogens and their immune response to an experimental challenge with a field strain of infectious bronchitis was evaluated by ELISA and production parameters were recorded. Results: Mortality was only observed in the control group and was 10%. All birds from the P, P&AO and AO groups responded to the challenge as would be expected of appropriately immunised chickens. Conclusion: The obtained results indicated that supplementation with synbiotic products and vitamins can enhance broiler chicken immunity and result in better production parameters.

West Nile Virus Lineage 2 in Free-Living Corvus cornix Birds in Poland.

The summer temperatures recorded in Poland in 2022 were among the highest in over 30 years and, combined with higher-than-expected rainfall, gave the impression of an almost tropical climate. Such climatic conditions were ideal for the transmission of vector-borne zoonotic diseases such as West Nile fever. In northeastern Poland, in the Mazowieckie region, the Polish event-based surveillance network reported increased fatalities of free-living hooded crows (Corvus corone cornix). West Nile virus (WNV) lineage 2 was identified for the first time as the etiological agent responsible for the death of the birds. WNV was detected in 17 out of the 99 (17.17%) free-living birds tested in this study. All the WNV-infected dead birds were collected in the same area and were diagnosed in September by the NVRI and confirmed by the EURL for equine diseases, ANSES, in October 2022. Unnaturally high temperatures recorded in Poland in 2022 likely favored the infection and spread of the virus in the avian population. A nationwide alert and awareness raising of blood transfusion centers and hospitals was carried out to prevent human infections by WNV.

Molecular analysis Polish isolates of goose hemorrhagic polyomavirus from geese and free-living birds.

Goose haemorrhagic polyomavirus (GHPV) is the viral agent of hemorrhagic nephritis and enteritis of geese (HNEG), a lethal disease of goose. The study describes the results of a molecular analysis Polish isolates of GHPV from geese and free-living birds based on complete VP1 gene and VP2 gene sequences. The sequences were analyzed and aligned with different GHPV isolates sequences accessible in the GenBank database. This study indicates affiliation GHPV isolates from fee-living birds and GHPV isolates circulating in Polish goose flocks and around the world to the same genetic groups, which proves their evolutionary relationship and indicates the potential role of free-living birds as a source of infections for poultry.

Current epidemiological situation in the context of inclusion body hepatitis in poultry flocks in Poland.

The research has been undertaken to understand the spreading of adenovirus strains in Poland's poultry flocks in the last six years. One hundred and forty-nine herds suspected of infection with adenoviruses were tested and the presence of poultry adenoviruses was found in 86 studied herds which were about 57,71% of examined flocks. Thirty-eight (44.18%) strains were connected with the infection of inclusion body hepatitis, 11 (12.79%) strains were isolated from digestive system dysfunction, 33 (38.37%) strains had been obtained from the flocks with no symptomatic changes/behaviour, and four (4.65%) strains were obtained from flocks with the manifestation of depression. Sequencing analysis was based on Loop L1 region of the HVR1-4 fragment of the hexon gene. The adenovirus strains were classified into five species FAdV-A-E, belonging to the following eight serotypes: FAdV-1/A, FAdV-5/B, FAdV-3/D, FAdV-2/11/D, FAdV-10/C, and FAdV-7/8a/E. The most common serotype in poultry turned out to be type/species FAdV-2/11/D, FAdV-5/B, and FAdV-7/8b/E while the least frequent was type/species FAdV-10/C (only two strains respectively of this type were isolated with the following range: FAdV-1/A 6 (6.97%), FAdV-5/B 24 (27,90%), FAdV-3/D 4 (4,65%), FAdV-10/C 2 (2,32%), FAdV-2/11/D 36 (41,86%), and FAdV-E 14 (16.27%). The understanding of genetic diversity, geographic distribution, and antigenic properties of fowl adenovirus strains (FAdVs) isolated in Poland have been evaluated.

Fowl adenovirus strains 1/A and 11/D isolated from birds with reovirus infection.

Inclusion body hepatitis (IBH) is, in some cases, a fatal disease affecting fowl by adenovirus strains which are subdivided into 5 species (A-E). In the current study, we investigated sequences from the Loop L1 region of the hexon gene of sequences of adenovirus field stains 1/A and 11/D isolated from a poultry flock co-infected with IBH and avian reoviruses ARVs. In early 2021, an epidemiologic survey highlighted the coinfection adenoviruses with other viruses (orthoreovirus infection) as being particularly deleterious within the poultry industry. Here, we investigated the Loop L1 HVR1-4 region of the hexon gene with relative synonymous codon usage (RSCU) designation and RSCU inclusive of all the mutations. These are the first results that have been presented on fowl adenovirus species A and D with simultaneous reovirus infection in 38-days old broiler chickens in Poland.

Characterisation of adenovirus strains represented species B and E isolated from broiler chicken flocks in eastern Poland.

Fowl adenovirus strains were isolated from the internal organs of 3-wk-old broiler flocks exhibited clinical signs associated with inclusion body hepatitis (IBH). The isolated strains were molecularly characterised and sequencing revealed three distinct clusters. One cluster showed close proximity at the nucleotide level with adenovirus type/species - 6/E, 7/E, 8a/E, and 8b/E. The second cluster contained five reference sequences belonging to the species FAdV-D and E. A third cluster contained one field and four reference sequences belonging to the FAdV-5/B, FAdV-4/C, FAdV-2/D, and FAdV-1/A type/species respectively. The heterogenicity, Relative Synonymous Codon Usage (RSCU), codon composition, and nucleotide frequencies were examined. Statistical analyses, were carried out. The maximum likelihoods for the examined sequences were estimated. The data indicated that correlation between isolated of adenovirus type/species 5/B, and E in Poland have been presented. Indicated adenovirus types and their combinations with locally circulating FAdVs strains could have implications for current detection methods and pathogenicity on infected chickens.

Serologic Survey for West Nile Virus in Wild Boars (Sus scrofa) in Poland.

Serum samples obtained from 474 wild boars (Sus scrofa) were collected from June 2017 to September 2018 from various areas of northern and southern Poland. Serum samples were examined by enzyme-linked immunosorbent assay. However, West Nile virus (WNV) antibodies were not detected. Previous studies on WNV in Poland have focused on experimental evidence and the presence of WNV antibodies in wild birds, horses, and humans, indicating a need for more surveys of domestic and wild mammals in Poland.

Occurrence of Marek's Disease in Poland on the Basis of Diagnostic Examination in 2015-2018.

INTRODUCTION: Marek's disease (MD) is a tumourous disease caused by Marek's disease virus (MDV) and most commonly described in poultry. The aim of the study was to determine the occurrence of Marek's disease virus infections in Poland and analyse clinical cases in the years 2015-2018. MATERIAL AND METHODS: The birds for diagnostic examination originated from 71 poultry flocks of various types of production. Birds were subjected to anatomopathological examination post mortem, during which liver and spleen sections and other pathologically changed internal organs were taken. These sections were homogenised with generally accepted methods, then total DNA was isolated and amplified with a real-time PCR. A pair of primers complementary to the MDV genome region encoding the meq gene were used. RESULTS: MDV infection was found predominantly in broiler chicken flocks (69.01%), and also in layer breeder (9.85%) and commercial layer flocks (7.04% each). CONCLUSION: The results of research conducted in the years 2015-2018 clearly indicate that the problem of MDV infections is still current.

Transmissible Viral Proventriculitis Caused by Chicken ProVentricular Necrosis Virus Displaying Serological Cross-Reactivity with IBDV.

Transmissible viral proventriculitis (TVP) of chickens is manifested in decreased body weight gains, poor feed conversion and weight diversity. Although TVP etiology has not been defined, a Birnaviridae family member, named chicken proventricular necrosis virus (CPNV) is considered as a potential factor of a disease. This study was undertaken in order to reproduce TVP and to evaluate its etiology. Broiler chickens of the TVP-infected group were inoculated with TVP positive proventriculi homogenate on the 24th day of life. Samples were collected, on infection day and 14 days post-infection (dpi). The 14 dpi anatomo- and histopathological evaluation, revealed that we have succeeded to reproduce TVP. TVP-infected birds gained 30.38% less body weight. In the TVP-infected group a seroconversion against picornaviruses, fowl adenoviruses (FAdV) and infectious bursal disease viruses (IBDV) was recorded with an ELISA test. Using RT-PCR and PCR, CPNV was detected in proventriculi and FAdV in spleens and livers of infected birds, 14 dpi. Our study supports that CPNV is involved in the development of TVP. We did not record the presence of IBDV in TVP or control birds, despite our recording of a seroconversion against IBDV in TVP infected birds. CPNV and IBDV belong to the same family, which allows us to assume serological cross-reactivity between them. The role of FAdV needs further evaluation.

Correction to: Detection of fowl adenovirus D strains in wild birds in Poland by Loop-Mediated Isothermal Amplification (LAMP).

An amendment to this paper has been published and can be accessed via the original article.

Isolation and molecular characterization of Fowl adenovirus strains in Black grouse: First reported case in Poland.

This article describes the isolation, molecular characterization, and genotyping of two fowl adenovirus (FAdVs) strains with GenBank Accession numbers (MT478054, JSN-G033-18-L and MT478055, JSN-G033-18-B) obtained from the internal organs of black grouse (Lyrurus tetrix). This study also reveals the first confirmation of fowl adenovirus in Poland, supporting one of the hypotheses about the probability of fowl adenovirus interspecies transmission. The adenovirus strain sequences were investigated via phylogenetic analysis and were found to have an overall mean pairwise distance of 2.189. The heterogeneity, Relative Synonymous Codon Usage (RSCU), codon composition, and nucleotide frequencies were examined. Statistical analyses and Tajima's test for the examined sequences were carried out. The Maximum Likelihood for the examined sequences substitutions was performed. The results of the sequence analysis identified MT478054, JSN-G033-18-L and MT478055, JSN-G033-18-B as strains of fowl adenovirus 2/11/D, with the Fowl adenovirus D complete sequence showing a 93% match. Wild birds may act as a natural reservoir for FAdVs and likely play an important role in the spreading of these viruses in the environment. The findings reported here suggest horizontal transmission within and between avian species.

Detection of fowl adenovirus D strains in wild birds in Poland by Loop-Mediated Isothermal Amplification (LAMP).

BACKGROUND: The present study on the role of strains of adenovirus in wildlife reservoirs, and their prevalence is under exploration. In several previous studies, the presence of adenovirus strains in wild birds has been investigated. Worldwide distribution and outbreaks of adenovirus infections have been reported by many authors. The present study investigated the prevalence of FAdVs in 317 samples of different bird species from the northwestern region of Poland. An applied specific, sensitive, and efficient, without cross-reactivity loop-mediated isothermal amplification (LAMP) method to gauge the prevalence of fowl adenovirus strains in wild birds was developed and used. RESULTS: The method was based on the sequence of the loop L1 HVR1-4 region of the hexon gene of the FAdV genome reference strains FAdV-2 KT862805 (ANJ02325), FAdV-3 KT862807 (ANJ02399) and FAdV-11 KC750784 (AGK29904). The results obtained by LAMP were confirmed by real-time PCR. Among 317 samples obtained from wild birds, eight FAdV isolates (2.52%) were identified and produced a cytopathic effect (CPE) in chicken embryo kidney cells (CEK). Three FAdV types belonging to species Fowl adenovirus D were detected, which were isolated from three adenovirus types 2/3/11, and have been confirmed in three mute swans (Cygnus olor), three wild ducks (Anas platyrhynchos), one owl (Strigiformes), and one common wood pigeon (Columba palumbus). CONCLUSIONS: This study provides the first accurate quantitative data for the replication of fowl adenovirus strains in wild birds in Poland, indicating adenovirus interspecies transmission, and demonstrating the circulation of FAdVs in wild birds.

Elaboration of Triplex PCR for Detection of Selected Viral Infections in Waterfowl.

INTRODUCTION: Viral infections are the greatest threat to waterfowl and cause significant economic losses. Diagnosis and differentiation of three goose viruses is difficult in the field and often requires laboratory confirmation. Therefore, the aim of the study was to develop a triplex PCR and optimise its parameters for simultaneous detection of DNA of goose parvovirus (GPV), goose polyomavirus (GHPV), and goose circovirus (GoCV). MATERIAL AND METHODS: The DNA of viruses isolated from field cases from the National Veterinary Research Institute's own collection was used for the study. The primer attachment temperature, the number of reaction cycles, and the Taq DNA polymerase and Mg2+ concentrations were optimised. The sensitivity and specificity of this triplex PCR was also determined. RESULTS: Based on the obtained results, triplex PCR parameters were optimised for simultaneous detection of DNA of GPV, GHPV, and GoCV in one sample. The following PCR products of the expected size were obtained: GPV DNA of 806 bp, GoCV DNA of 571 bp, and GHPV DNA of 180 bp. CONCLUSION: The developed triplex PCR method proved to be useful for simultaneous detection of infections with three waterfowl viruses and will be used in relevant laboratory diagnostics.

Deep analysis of Loop L1 HVRs1-4 region of the hexon gene of adenovirus field strains isolated in Poland.

BACKGROUND: To date, studies on loop L1 HVRs1-4 region of the hexon gene in fowl adenovirus genome (FAdVs) lack comprehensive molecular data. In this study detailed prospectively obtained sequences from field adenovirus strains, NVRI, Poland have been analyzed. METHODS: Overall hundred and thirty seven adenovirus strains were collected, evaluated, and examined of hyper variable loop L1 region HVRs1-4 of the hexon gene for the presence of similarity, mutations, tertiary structure, and spinal conformation. RESULTS: Sequences were characterized, and divided for five species and seven types, FAdV-A-E/FAdV-1/2/4/5/7/8a/8b/11. The presence of predicted tertiary structure depending on type/species were determined. Analysis of specific selected sequences: GQMTN 1/A, 7/E, and 8b/E, GQMTT 2/11/D, GQLSN 4/C, GQMTH 5/B, and GQMSN 8a/E in examined HVRs1-4 Loop L1 region of hexon gene compared to tertiary structure indicated that this visibly conservative region represents the antigenic binging activity. CONCLUSION: This is the first molecular study on tertiary structure on HVRs1-4 region in adenovirus genome conducted in Poland. Analysis indicated specific sequence in Loop L1 HVR1-4 region which is strictly responsible for antibodies binding. This information could assist during the process connected with specific preventive strategies based on their molecular genome investigation and new facilitate studies. This study will help to better understand the mechanisms of pathogenicity of adenovirus strains provide a guide for disease control in birds.

A comparative pathogenicity analysis of two adenovirus strains, 1/A and 8a/E, isolated from poultry in Poland.

Fowl adenoviruses (FAdVs) are the causative agents of multietiological syndromes and diseases in poultry flocks. During a routine diagnostic examination, two FAdVs strains were isolated. Molecular typing of these isolates based on the partial loop L1 HVR1-4 region of the hexon gene sequence revealed the presence of different FAdV isolates: 1/A-61/11z (GenBank accession number KX247012, APP94082), and 8a/E-6/12j (GenBank accession number: KP890032, ALB00550), and comparative genome analysis indicated small differences between these two viruses. The next step of the study was the estimation of the pathogenicity of these isolates in specific-pathogen-free (SPF) chickens. Chickens were divided into three groups, with 20 chickens per group infected intraperitoneally on the first day after hatching. Group I consisted of chickens infected with strain FAdV-1/A-61/11z, group II consisted of chickens infected with strain FAdV-8a/E-6/12j, and group III consisted of uninfected birds. Clinical signs observed in infected chickens included poor growth, apathy, prostration, ruffled feathers, crouching position, and huddling behavior. The mortality rate in chickens infected with FAdV-1/A-61/11z was 10% at 10 days postinfection (dpi), and no mortality was observed in chickens infected with the FAdV-8a-6/12j strain. The mean real-time PCR threshold cycle (Ct) value was 39.70%. The detection limit of these assays was 8 copies, with an efficiency of 91.03% and 95.17% and regression square (R2) values of 0.991 and 0.997, respectively, with a mean pathogen load of 4.8 × 106.0 copies/µl. The assays did not demonstrate cross-reactivity between types 1/A and 8a/E and non-targeted poultry viruses. Adenoviral DNA was detected in the liver, spleen, kidney, gizzard, intestine, bursa of Fabricius, and thymus of every examined dead and euthanized chicken from groups I and II between the third and fourth week postinfection. This is the first study conducted on the pathogenic and apathogenic strains FAdV-1/A and FAdV-8a/E, showing the presence of the virus in multiple tissues in chickens in Poland. This study revealed that it is very likely that the FAdV-1/A-61/11z strain is able to cause clinical inclusion body hepatitis (IBH) in chickens and that it is slightly more virulent than the FAdV-8a/E-6/12j strain, although both are primary pathogens of the disease.

Occurrence of Reovirus (ARV) Infections in Poultry Flocks in Poland in 2010-2017.

INTRODUCTION: Avian reovirus (ARV) infections in poultry populations are reported worldwide. The reovirus belongs to the genus Orthoreovirus, family Reoviridae. The aim of the study was to evaluate the incidence of ARV infections in the poultry population based on diagnostic tests performed in 2010-2017. MATERIAL AND METHODS: Samples of the liver and spleen were collected from sick birds suspected of ARV infection and sent for diagnostics. Isolation was performed in 5-7-day-old SPF chicken embryos infected into the yolk sac with homogenates of internal organs of sick birds. Four primer pairs were used to detect the σNS, σC, σA, and µA ARV RNA gene fragments. A nested PCR was used for the detection of the σNS and σC genes. RESULTS: In 2010-2017, ARV infection was found in birds from 81 flocks of broiler chickens and/or layers, 8 flocks of slaughter turkeys, and in 4 hatchery embryos at 17-20 days of incubation. The primers used in RT-PCR and nested PCR did not allow effective detection of ARV RNA in all virus-positive samples. CONCLUSION: The problem of ARV infections in the poultry population in Poland still persist. The primers used for various ARV segments in RT-PCR and nested PCR did not allow effective detection of RNA in the visceral organs of sick birds. The presented results confirm the necessity of using classical diagnostic methods (isolation in chicken embryos, AGID).

Avian Poxvirus Infection in Polish Great Tits (Parus Major).

INTRODUCTION: Avian poxvirus infections are widespread in the domestic poultry population but are also reported in wild birds. In poultry, these infections cause significant economic losses, while wild birds may be a reservoir for poxvirus which affects breeding poultry. However, wild birds may also exhibit characteristic anatomopathological changes. This study concerns the infection of wild-living great tits (Parus major) with the avian poxvirus in Poland. MATERIAL AND METHODS: Samples of internal organs and skin collected from great tits were homogenised and total cellular DNA was isolated. In PCR, the primers complementary to gene encoding the core protein 4b of the HP44 strain of fowl poxvirus (FPV) were used. RESULTS: After electrophoresis in 2% agarose gel, the PCR product of 578 bp characteristic for FPV was obtained in DNA samples isolated from skin lesions and the heart. The analysis of the nucleotide sequence of the virus strain showed 99% similarity to many poxviruses previously isolated from great tits and other free birds at various sites in the world. CONCLUSIONS: This paper is the first clinically documented evidence obtained in laboratory conditions of avian poxvirus cases in great tits in Poland.

Molecular characterisation of fowl adenovirus type 7 isolated from poultry associated with inclusion body hepatitis in Poland.

The fowl adenovirus field strain FAdV-JSN-5/10j (GenBank accession number KP879219) was isolated from the intestine of a 7-week-old chicken diagnosed with inclusion body hepatitis and simultaneously with Marek's disease, and for that reason, it was chosen for molecular study. It was identified as fowl adenovirus genotype 7 (species Fowl aviadenovirus E) based on nucleotide sequence analysis of the loop L1 region of the hexon gene. Nucleotide sequence alignment of this strain, FAdV-7 reference strains B-3A ATCC VR-832 (AF339922) and YR36 (AF508955), and eight additional FAdV-7 field strains confirmed its classification as FAdV-JS-5/10j and showed that these viruses are very similar to each other. Additionally, we described mutations and their influence on the amino acid sequence, nucleotide composition, and relative synonymous codon usage. Immunofluorescence of cell cultures infected with 104.5 TCID 50 per 0.1-ml dose of the FAdV-JSN-5/10j strain demonstrated the presence of a cytopathic effect. Infection of fowl with adenoviruses raises concerns for poultry production, and thus, the efficient detection of adenovirus infection is crucial. This is the first attempt to describe the molecular characteristics of FadV-7 strains isolated in Poland.

Whole genome sequencing of Fowl aviadenovirus A - a causative agent of gizzard erosion and ulceration, in adult laying hens.

Gizzard erosion and ulceration (GEU) caused by fowl aviadenovirus serotype 1 (FAdV-1) of the species Fowl aviadenovirus A (FAdV-A) represents an economically important problem in poultry production. The disease affects mostly young chicken broilers or layers before production. In this study, an unusual GEU outbreak in a flock of laying hens at 38weeks of age is described. The affected flock showed elevated mortality rates, with the highest number of dead birds appearing between the 39th and 40th week of life, with a subsequent reduction in laying performance and decreased total egg weight. Post-mortem examination showed the presence of erosion in multiple areas of the gizzard, with wall perforation in the proximity of the interventriculus. FAdV antibodies were detected in all examined sera with an ELISA assay. The virus was isolated from pathologically altered gizzards. PCR, subsequent sequencing and phylogenetic analysis of the partial hexon gene confirmed the presence of FAdV-A DNA. To investigate the molecular background of FAdV-A which causes GEU in adult hens, whole genome sequencing was performed on two FAdV-A strains - strain W-15, obtained from the outbreak described in this study and strain 61/11z, isolated from a GEU outbreak in 3-week-old broiler chickens in 2011. The genome size of FAdV-A W-15 is 43,849bp. Genome sequence and genome organization resembles those of the reference, apathogenic CELO strain and the newly sequenced GEU strain, 61/11z. Most amino acid changes, between CELO and GEU strains, were observed in ORF0, ORF1, ORF14, IVa2, polymerase, pIIIa, penton base and fiber-2. Analysis conducted on the translated ORFs revealed that W-15 and 61/11z are nearly identical, with the highest rate of amino acid mutations in pTP, 100K, ORF9 and ORF10. In this study, the occurrence of GEU, caused by FAdV-1 infection, in adult layer chickens and the effects of such infection on egg production parameters are described in detail. Moreover, the whole genome sequences of two pathogenic, GEU inducing FAdV-A strains have been provided and characterized for the first time, which in the future will help to pinpoint the viral factors involved in pathogenicity.