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Journal of Avian Research RESEARCH ARTICLE open access Effect of lentogenic Newcastle disease virus (Lasota) on low pathogenic avian influenza virus (H9N2) infection in fayoumi chicken 1 Sajid Umar , Tamoor Azeem2*, Salman Ahmed Abid2, Aqsa Mushtaq3, Kiran Aqil3, Muhammad Rizwan Qayyum3, Abdul Rehman4 1 National Veterinary school Toulouse, France, 2 Department of Pathology, University of Veterinary and Animal Sciences, Lahore, Pakistan. 3 Veterinary Research Institute, Lahore, Pakistan. 4Friedrich-Loeffler-Institute of Epidemiology Berlin, Germany Abstract: Low pathogenicity avian influenza virus (LPAIV) and lentogenic Newcastle disease virus (lNDV) are are two of the most economically important viruses affecting poultry worldwide. Co-infections usually occur but cannot be easily diagnosed due to confusing similar clinical signs. Fayoumi is indigenous chicken of Pakistan on which the impact of co-infections is still unknown. The objective of this study was to investigate the effect of lNDV on the infectivity and excretion of LPAIV in fayoumi chicken. Four week old fayoumi chicks were inoculated intranasally with 106 median embryo infectious of lNDV vaccine strain (LaSota) and a H9N2 LPAIV (A/Chicken/Pakistan/UDL/08 H9N2) simultaneously. No clinical signs were observed in chickens infected with the lNDV. All chicken showed mild to moderate respiratory distress with LPAIV alone or in combination with lNDV. Clinical and necropsy findings revealed non synergistic behaviour of two viruses for the development of clinical signs and lesions. However, the pattern of virus shed was different with co-infected chickens, which excreted lower titres of lNDV and LPAIV at first three days post inoculation (dpi) as compared to singly inoculated chicken but after 3 dpi co-infection resulted in significantly higher number of oropharangeal and cloacal swabs detected positive for LPAIV and lower number for lNDV. The knowledge obtained from the study serves the dual purpose of shedding light on the different replication behaviours of LPAIV in early days of experiment which may be due to competition for receptor binding with lNDV, as well as the more pathogenic behaviour of LPAIV (H9N2) in fayoumi chickens of Pakistan. Key words: Poultry, LPAIV, NDV, co-infection Citation: Umar S, Azeem T, Abid AS, Mushtaq A, Kiran A, Qayyum MR, Rehman A. Effect of lentogenic Newcastle disease virus (Lasota) on low pathogenic avian influenza virus (H9N2) infection in fayoumi chicken. J Avian Res, 2015, 1(1):1-4 Received: Jan 01, 2015; Revised: Feb 28, 2015; Accepted: Mar 11, 2015 Copyright: 2015 Umar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Competing Interests: The authors have declared that no competing interests exist. *Corresponding author: Tamoor Azeem; Email: [email protected] Introduction Newcastle disease virus (NDV) and Avian influenza virus (AIV) have been great threat to poultry industry and have caused great economical losses by decreasing egg and meat production. These viruses originated from wild birds and then transmitted to domestic poultry birds producing mild to severe clinical infections of respiratory tract. Both of these are negative-sense single stranded, RNA viruses. NDV belong to Paramyxoviridae family and also known as avian Paramyxovirus. Based on virulance and pattern of sequences present near protease cleavage site of fusion (F) protein NDV have been catogaorized into different pathotypes producing diseases of different types and severity in chicken, those are viscerotropic velogenic, neurotropic velogenic, mesogenic, lentogenic or asymptomatic [1]. AIV are member of Orthomyxoviridae family and also known as orthomyxovirus. Avian influenza viruses are classified as low pathogenic (LP) and high pathogenic (HP) viruses depending upon the presence of multiple basic amino acids at cleavage site of the HA precursor protein and virulence in chicken [2]. Diseases produced by virulent strains of NDV (velogenic and mesogenic) and highly pathogenic avian influenza (HPAI) viruses are notifiable to the World Organization for Animal Health [3]. Lentogenic strains of NDV are mostly used as live vaccines for the protection virulent Newcastle disease virus (vNDV) infections in poultry. These lentogenic or vaccine strains of NDV are known to cause little or mild respiratory distress in specific pathogen free (SPF) chickens under experimental conditions [1]. However, during field outbreaks they have been reported to cause severe respiratory distress leading to decrease production of eggs and meat especially when joined by other respiratory pathogens or immunosuppressive agents of the environment. Lasota and B1 are the most commonly used strains of NDV which are used in live and killed lNDV vaccines for the prevention of vNDV outbreaks in developed and developing countries [1]. Similarly, LPAIVs produce very mild respiratory infections in SPF experimental chickens but co-infections with other pathogens including viruses can exacerbate disease outcomes under field conditions leading to severe respiratory problem. [4] LPAI infection is an emerging threat to most of the countries especially Middle East and Asian countries [5]. Low pathogenic avian influenza virus (H9N2), circulating in Pakistan has been declared to have novel genotype. However, vaccines for the prevention of LPAI are not used routinely and only inactivated and vectored vaccines are allowed to be used against LPAI. Coinfections LPAIV and lNDV commonly occur in field and present a complicated clinical picture of mixed clinical signs and lesions which often leads to misdiagnosis of these viruses [6,7].To date very little is known about the co-infections of these two viruses especially in indigenous chicken. Virus and bacteria co-infections have been reported with increased clinical signs and lesions as compared to single infection in chicken [8,9] Conversely, infection of a host with one virus may affect infection by a second virus, a phenomenon explained by the occurrence of viral interference, in which cells infected by a virus do not permit multiplication of a second virus [10]. Measurable differences may include changes in tissue permissiveness or tropism, viral replication, patterns of virus progeny production and release, latency, pathology including immunopathology, and immunological responses [11]. In addition, viral interference may be detrimental to detecting viruses in co-infected flocks since lower or undetectable virus titers might fail to give a complete diagnosis [12]. Exposure to lNDV, either as live vaccines or field strains, is nearly unavoidable for commercial and non-commercial poultry worldwide, and co- 31 J Avian Res, 2015, 1(1):1-5 infections with LPAIV are likely to occur. Both viruses replicate in epithelial cells of the respiratory and intestinal tracts, where there are trypsin-like enzymes, likely competing for target cells or replicating in adjacent cells. Whether co-infections with LPAIV and lNDV will exacerbate clinical signs of disease in infected birds or produce viral interference, masking infections by one or other virus, is unknown. In this study we examined the effect of co-infections of fayoumi chickens with LaSota lNDV vaccine strain and a LPAIV (A/Chicken/Pakistan/UDL/08 H9N2) by inoculating the viruses simultaneously and determining differences in pathogenesis (clinical signs, lesions), presence of the viruses in tissues, duration and titer of virus shedding and Seroconversion to both viruses. Such a study design replicates field situations in countries free of virulent NDV, but with active NDV vaccination programs and where LPAI outbreaks periodically occur [1, 5]. Materials & methods Virus The UDL-01/08 H9N2 virus was a field isolate obtained from Quality operation laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan. Viral stocks were prepared and titrated in 9-day-old to 10-day-old specific pathogen free embryonated chicken eggs [13]; the median embryo infectious dose (EID50) was calculated using previously reported methods [14]. The viral stocks were diluted in sterile brain–heart infusion medium containing antimicrobials to yield a final titre of 106 EID50/0.1 ml [15]. Birds Experimental study protocol was approved by the Animal care and research committee of the University of Veterinary and Animals Sciences, Lahore and experimentation was carried out according to the guidelines of committee. Three-week-old fayoumi chicks (indigenous layer hens), were acquired from a local supplier. Blood and buccal swab samples were taken from all birds and analysed by haemagglutination inhibition (HI) and virus isolation (VI) in eggs using standard methods [16] to ensure that the birds were serologically naïve and free from influenza virus and Newcastle disease virus infection prior to the start of the experiment. Each group of birds was housed separately in cages in separate rooms (Table 1).The birds were acclimatized for 7 days prior to inoculation. Feed and water were provided ad libitum. All treatment groups contained 10 birds and were inoculated by the intranasal routes with a dose of 106 EID50 of each virus or sham inoculum. The viruses were given alone and in combination simultaneously on the same day. The birds were monitored three times daily for clinical signs (reluctance to move, anorexia, congestion of eyes, respiratory signs mainly sneezing, swollen head, haemorrhage on shanks Blood samples for serology were taken before virus inoculation (day 0) and 14 days post inoculation (dpi). Oropharyngeal (OP) and cloacal (CL) swabs were collected from all birds from 1 to 7 dpi to assess virus shedding. Three birds from each group were euthanized at 3 dpi for necropsy findings and tissues were collected in 10% neutral buffered formalin to evaluate microscopic lesions and the extent of virus replication in tissues as described previously [15, 18]. At 14 dpi remaining birds were bled for serology and euthanized by the intravenous (IV) administration of sodium pentobarbital (100 mg/kg body weight). RNA extraction and Real-Time PCR Oropharyngeal and cloacal swabs were collected in 2 mL of BHI broth with a final concentration of gentamicin (200 μg/mL), penicillin G (2000 units/mL), and amphotericin B (4 μg/mL) and kept frozen at −70 °C until processed. RNA was extracted using the QIAamp viral RNA isolation kit. Quantitative real time RT-PCR (qRT-PCR) for AIV and Newcastle disease virus (NDV) detection was performed as previously described [19] with modifications. qRT-PCR reactions targeting the influenza virus M gene [20] and NDV M gene [21] were conducted using Quanti-fast SYBR green RT-PCR one step RT-PCR Kit (Qiagen) and the Light cycler 480, Real-Time PCR system (Roch Life sciences Switzerland). The RT step conditions for both primer sets were 10 min at 45 °C and 95 °C for 10 min. The cycling conditions for AIV were 45 cycles of 15 s, 95 ° 45 s, 60 °C; and for NDV were 40 cycles of 10 s, 94 °C; 30 s, 56 °C; 10 s, 72 °C. The calculated qRT-PCR lower detection limit was for AIV was 100.5RNA copies (log10)/mL and 100.6 RNA copies (log10)/mL for NDV. A standard curve for virus quantification was established with RNA extracted from dilutions of the same titrated stock of the challenge virus, and results also reported as Log10 RNA copies/mL equivalents Serology: Hemagglutination inhibition (HI) assays were performed to quantify antibody responses to virus infection as previously described [3] with serum collected from birds at 14 dpi. Titers were calculated as the highest reciprocal serum dilution providing complete haemagglutination inhibition. Serum titers of 1:8 [20] or lower were considered negative for antibodies against AIV or NDV Statistical analysis Data were analyzed using Prism v.5.01 software (GraphPad Software Inc. La Jolla, CA, USA) and values are expressed as the mean ± standard deviation of the mean (SDM). One-way ANOVA with Tukey post-test was used to analyze HI titers. The number of birds shedding virus were tested for statistical significance using Fisher’s exact test. Two-way ANOVA with Bonferroni multiple comparison analysis was used to evaluate virus titers in swabs. For statistical purposes, all qRT-PCR negative oropharyngeal and cloacal swabs were given a numeric value of 100.5RNA copies (log10)/mL for LAIV and 100.6 RNA copies (log10)/mL for lNDV. All HI-negative serum was given a value of 3 log2. These values represent the lowest detectable level of virus and antibodies in these samples based on the methods used. Statistical significance was set at p < 0.05 unless otherwise stated. Results Clinical signs No clinical signs were observed in chicken exposed to lNDV alone. However, all chicken exposed to LPAIV, regardless of lNDV exposure, presented mild to moderate clinical signs consisting of periocular edema, conjunctivitis, sinusitis, ruffled feathers, and lethargy. These clinical signs were first observed at 3 dpi and lasted until 7 dpi. No differences in the severity of the clinical signs were observed between the groups inoculated with both LPAIV and lNDV and the group inoculated only with LPAIV. Virus shedding All birds were negative for H9N2 AIV by serology and virus isolation in eggs prior to inoculation. All birds in inoculated groups became infected with H9N2 AIV as determined by detection of the H9N2 AIV matrix gene in OP and CL swabs. The total number of positive swabs and the duration of virus shedding varied among different groups (Table 3). LPAIV viral shedding was mainly through the oropharyngeal (OP) route (p<0.05). At early time points (1–3 dpi), co-infected birds presented lower amounts of viral shedding than birds receiving lNDV or H9N2 virus alone. These differences were significant at 1 and 3 dpi when chickens coinfected with LPAIV and lNDV showed significantly lower viral titers compared to single lNDV or H9N2 virus infected birds (p<0.05). The peak H9N2 AIV shedding in OP swabs varied between groups as determined by RT PCR results (Fig. 1 & 2). Birds in the lNDV + H9N2 group had peaks for AIV OP shedding at 5dpi. Similarly, peak of lNDV OP shedding was at 5dpi in lNDV group. Moreover, virus shedding period was longer in birds of the lNDV+H9N2 group than those in the H9N2 group. Lower virus titer of lNDV was observed in co-infected group than lNDV infected group. Necropsy and histopathological findings No gross lesions were observed in any of the birds necropsied at 3 dpi, except for the chicken inoculated with LPAIV alone or in combination with lNDV, which had mild conjunctivitis, sinusitis, enteritis and moderate tracheitis and airsaculitis. The microscopic lesions observed were consistent with gross lesions of LPAIV and lNDV infection (Table 2). Lesions present in tissues from LPAIVinfected chicken included mild rhinitis, sinusitis and enteritis characterized by mild accumulation of inflammatory cells and exudate while moderate airsaculitis and tracheitis having moderate accumulation inflammatory cells and exudate. No significant difference in the severity of lesions was found between chicken infected only with LPAIV and chicken co-infected with LPAIV and lNDV. Serology HI assays were used to test for antibodies against LPAIV and lNDV (Table 4). All chickens seroconverted to both LPAIV and lNDV, among the inoculated groups. However, a clear difference was found in lNDV and LPAIV titers in chicken exposed to alone than 2 J Avian Res, 2015, 1(1):1-5 Table. 1: Experimental design Groups Age (days) Number of birds A B C 28 28 28 10 10 10 D 28 10 Route of inoculation Viral strain Dose per bird Negative control (non inoculated) Lasota vaccine strain (A/Pakistan/chicken/UDL-01/08) (A/Pakistan/chicken/UDL-01/08)+ Lasota vaccine strain 0.5ml normal saline 106 EID50/0.5 ml lNDV Lasota strain 106 EID50/0.5 ml LPAIV 106 EID50/0.5 ml LPAIV 106 EID50/0.5 ml lNDV IN IN IN IN Table. 2: Microscopic lesions at day 3 PI Groups Rhinitis/sinusitis tracheitis bronchitis A _ _ _ B + C + ++ + D + ++ + Notes: Whereas, -no lesions, + mild , ++ moderate , +++ severe pnuomonitis _ + + Air sacculitis _ ++ ++ enteritis _ + + Table. 3: Infection and viral sheddinga (Viral sheddinga of LPAIV detected in OP and CL swabs after single infection or co-infection with lNDV and H9N2 LPAIV in Fayoumi chicken). Swabs OP Groups B C D CL B C D 1 (lNDV) (LPAIV) LPAIV lNDV (lNDV) (LPAIV) LPAIV lNDV d 4/7 5/7 2/7 3/7 2/7d 3/7 2/7 4/7 2 3 Days post-inoculation 4 5 6 7 8 9 10 Totalb meanc 2/7 3/7 3/7 4/7 3/7 4/7 3/7 6/7 6/7 7/7 5/7 7/7 4/7 5/7 2/7 7/7 7/7 7/7 7/7 5/7 7/7 5/7 6/7 3/7 0/7 0/7 0/7 0/7 0/7 1/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 25 35 27 23 20 30 26 24 3.57 5 3.85 3.2 2.8 4.2 3.7 3.4 4/7 7/7 7/7 3/7 3/7 7/7 7/7 3/7 2/7 4/7 2/7 1/7 1/7 3/7 4/7 1/7 0/7 2/7 1/7 0/7 0/7 2/7 2/7 0/7 a Determined by rRT-PCR. b Total number of positive swabs. c Mean number of viral shedding days. d Number of positive birds/total number of birds (n = 7 indicates the number of birds that were sampled during the entire study period, excluding the birds that were euthanized in the first 3 days post single infection or co-infection combined infections, indicating that the presence of LPAIV might be interfering with the production of antibodies against lNDV. These results suggest that infection with a heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated, which decreases with time. Discussion Co-infections of different viruses and bacteria especially lNDV and LPAI are expected to occur and have been reported in poultry [22,23], but the impact of such co-infections on several host responses including clinical outcome, viral shedding dynamics, seroconversion, and sites of virus replication in fayoumi chickens is unknown. Co-infections of AIV and NDV have been studied in vitro using cell cultures or chicken embryos, and interference between these viruses has been reported, with one virus inhibiting the growth of the other [7,24, 25] In contrast to in vitro or in ovo studies, in vivo experiments examine the overall effect of coinfections by incorporating the complexity of the whole organism, including different target cells and immune responses. In our coinfection studies, all chickens became infected with lNDV and LPAIV as shown by virus shedding results, and a significant reduction in virus replication was observed for first 3 days when birds were co-infected versus single virus infected. In spite of the differences in virus replication, co-infection of LPAIV and lNDV had no effect on the severity of clinical signs. Typically, chickens lack clinical signs in experimental infection with LPAIV or lNDV, which was corroborated by mild microscopic lesions. Lack of clinical signs was also observed in chicken infected with lNDV alone. However, all chicken infected with LPAIV, coinfected or not, showed mild transient upper respiratory signs and moderate inflammation and necrosis in the epithelia of nasal trachea and air sacs accompanied by mild exudate. These results are consistent with previous reports mentioning similar findings [26, 27]. In addition, this H9N2 LPAIV strain is known to be chicken adapted, with a very low mean bird infectious dose required to produce infection [28]. Therefore, the host species is a factor that can influence the severity of clinical signs and amount of virus replication in such virus co-infections. In this study, an effect in the pattern of viral shedding was also found in the fayoumi chickens, indicating that virus interference can occur, but to a lesser degree, as long as there is viral replication. We do expect similar results with other viruses. In this study, viral shedding patterns were clearly affected in chickens exposed to lNDV and LPAIV simultaneously. Although LPAIV OP shedding was significantly less when measured at 3 dpi but peak virus shedding time was same for all groups. Likewise, co-infection of chickens with LPAIV also affected lNDV OP virus shedding, with initial lower virus replication in co-infected birds than birds receiving only LPAIV, but higher and more prolonged virus shedding at later time points. The presence of high virus titers was associated with clinical signs and microscopic lesions in respiratory tissues of all chicken infected with LPAIV. lNDV OP virus shedding was clearly affected by LPAIV replication, with fewer co-infected birds shedding lNDV and with significantly lower lNDV titers than chicken infected with lNDV alone. However, lNDV replication increased slowly in birds that received LPAIV probably because by then the effect of LPAIV replication had diminished. This suggests that infection with a heterologous virus may result in temporary competition for cell receptors or susceptible cells, resulting in decreased initial replication of the second virus; but as replication of the first virus declines, the second virus increases to fill the gap. Viral interference is a phenomenon in which a cell infected by a virus does not permit multiplication of a second homologous or heterologous superinfectant virus [10]. Viral interference can be explained by different mechanisms including: competing by attachment interference therefore reducing or blocking of receptor sites for the super-infecting virus; competing intracellularly for replication host machinery; and virus-induced interferon interference [29]. lNDV and LPAIV replicate in cells where there are trypsin-like enzymes such as in the upper respiratory and intestinal epithelia [4,30] and might compete for the same target cells or replicate in adjacent cells. The LaSota virus, as a lNDV, 1 J Avian Res, 2015, 1(1):1-5 binds through the HN glycoprotein to sialic acid-containing receptors on cell surface, as well as the HA glycoprotein does for LPAIV [31]. Replication of one virus might also be affected by previous replication in the same site of another virus that has already activated antiviral immune responses including Table. 4: Serological status for LPAIVand lNDV, as determined by HI test, of fayoumi chicken at before inoculation ( day 0) and after inoculation (14 days) in single infection and co-infection groups. HI titre results a Day 0 Day 14 B 7/7b (HI<8) 7/7 (256) C 7/7 (HI<8) 7/7 (512) lNDV 7/7 (HI<8) 7/7 (128) D LPAIV 7/7 (HI<8) 7/7(512) a Number of positive birds/total (HI titre). b Number of positive birds/total number of birds (n = 7 indicates the number of birds that were sampled during the entire study period, excluding the birds that were euthanized in the first 3 days post single infection or co-infection) Groups immunomodulators or recruitment of immune cells. Although the LaSota lNDV strain is known to be a weak interferon inducer as part of their low virulent phenotype profile [32] local interferon production might still be able to interfere with LPAIV replication. In fact, previous studies in embryonating eggs showed that LaSota lNDV could suppress growth of a H9N2 LPAIV’s, if given prior to the LPAIV [24]. Influenza viruses also induce interferon [33,34], which could have been one mechanism by which the high LPAIV replication in the turkeys inhibited lNDV replication. Viral interference has also been suggested in other studies with influenza virus such as the pandemic H1N1 when it was shown that an increase in the proportion and number of rhinovirus diagnoses in humans occurred in parallel with the decrease of influenza diagnoses, suggesting that rhinoviruses inversely affected the spread of the pandemic H1N1 virus [35, 36]. Fig. 1: Mean virus titre values (log10 RNA copies/ml) of lNDV detected in OP and CL swabs/day post inoculation in group B and D Fig. 2: Mean Virus titre values (log10 RNA copies/ml ) of LPAI detected in OP and CL swabs/day post inoculation in group C and D Experimental in vivo co-infections of NDV and LPAIV are scarce in chickens. França et al. [37] performed co-infection in wild ducks with LPAIV and lentogenic NDV wild bird strains and observed differences in the pattern of virus shedding depending on the time of co-infection. The authors suggest that competition for replication sites and/or differences in fitness for replication may explain the effects of co-infection with lentogentic lNDV and LPAIV. Other studies have examined co-infection of LPAIV and lNDV with other respiratory viruses of poultry. Research has shown that infectious bronchitis virus (IBV) interfered with the replication of lNDV [38, 39] However, IBV live vaccine increased the severity of H9N2 LPAIV infections [11, 12], and it was suggested that IBV was a supplier of trypsin-like proteases therefore enhancing the reach of systemic sites by the virus. In our case, such an exacerbation would not occur, since neither the LaSota lNDV strain, nor the LPAIV can provide extra enzymatic activity to each other. In other studies, coinfection of turkeys with lNDV and another respiratory virus, avian pneumovirus (APV), induced more severe disease compared to turkeys infected with APV or NDV [40-42], and dual vaccination of turkeys with lNDV and hemorrhagic enteritis virus (HEV) live vaccines enhanced the pathologic response of the host [16]. In chickens, although the co-infection with LPAIV and lNDV interfered with viral replication as seen in the viral shedding patterns, the reduction in the humoral immune response was not observed, since all chickens seroconverted with similar antibody titers to LPAIV and NDV, regardless if they were co-infected or not. This is similar to what has been reported with experimental coinfections with IBV and live lNDV and some other diseases vaccines in broilers [39]. Although no significant effect of coinfection was observed on HI titers with these particular viruses, an effect might be seen in chickens infected with more virulent strains of NDV and AIV. [22]. Our co-infection study was performed under controlled conditions in order to examine the specific interactions between the two viruses when given at high challenge doses. This might not be representative of what happens under field conditions where poultry are exposed to many viruses and other infectious and non-infectious disease agents. However, the results obtained underline the importance of co-infections which can either exacerbate clinical disease, or, like in our study, affect virus replication by lowering viral titers to under the levels of detection and affecting serological results, and in some cases increasing the time virus was shed which could favor prolonged transmission. In addition, exposure to lower challenge doses of these viruses in the field could also affect the results of co-infection. The effects of virus co-infection will most likely vary depending on how well adapted the viruses are to a specific bird species, on the virulence of the viruses involved, on the timing of co-infections, and on other concomitant infectious and environmental factors. Evaluating the infectious status in birds might be necessary when developing vaccination protocols using live attenuated vaccines. The role of viral interference in the spread of AIV and NDV needs further examination as also the role of co-infections in terms of altering the severity of clinical signs and lesions. The identification of factors that influence co-infection interference or elements that favor a delay in infection of one virus at expense of another virus will provide new insights in the pathogenesis of these viruses, allowing better development of new diagnostic and vaccine technologies for prevention and control of these infections. References 1. 2. 3. 4. 5. 6. Alexander DJ, DA Senne. Newcastle disease. In: Diseases of Poultry. 12th edition. Edited by Saif YM, Glisson JR, McDougald LR, Nolan LK, Swayne DE. Ames, Iowa, USA: Blackwell Publishing 2008; pp: 75–100. Suarez DL. Influenza A virus. In: Avian Influenza. Edited by Swayne DE Blackweel Publishing. Ames, Iowa, USA 2008; pp: 3–22. OIE: Avian influenza. In: Manual for Diagnostic Tests and Vaccines for Terrestrial Animals. 2012. Swayne DE, DA Halvorson. Influenza. In: Diseases of Poultry. 12th edition. Edited by Saif YM, Glisson JR, McDougald LR, Nolan LK, Swayne DE. Blackwell Publishing, Ames, Iowa, USA, 2008; pp: 153–184. Halvorson DA. Control of low pathogenicity avian influenza. In: Avian influenza. Edited by Swayne DE.: Blackwell Publishing, Ames Iova, USA 2008; pp: 513–536. El Zowalaty ME, M Abin, Y Chander, PT Redig, SM Goyal. Isolation of H5 avian influenza viruses from waterfowl in the 5 J Avian Res, 2015, 1(1):1-5 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. upper Midwest region of the United States. Avian Dis 2011; 55: 259–262. Shortridge KF, King AP. Cocultivation of avian orthomyxoviruses and paramyxoviruses in embryonated eggs: implications for surveillance studies. Appl Environ Microbiol 1983; 45: 463–467. Stipkovits L, Egyed L, Palfi V, Beres A, Pitlik E, Somogyi M, Szathmary S, Denes B. Effect of low-pathogenicity influenza 4 virus H3N8 infection on Mycoplasma gallisepticum infection of chickens. Avian Pathol 2012; 41:51–57. Pan Q, Liu A, Zhang F, Ling Y, Ou C, Hou N, He N. Coinfection of broilers with Ornithobacterium rhinotracheale and H9N2 avian influenza virus. BMC Vet Res 2012; 8: 104. Dianzani F. Viral interference and interferon. Ric Clin Lab 1975; 5: 196–213. DaPalma T, BP Doonan, NM Trager, LM Kasman. A systematic approach to virus-virus interactions. Virus Res 2010; 149:1–9. El Zowalaty ME, Chander Y, Redig PT, Abd El Latif HK, El Sayed MA, Goyal SM. Selective isolation of avian influenza virus (AIV) from cloacal samples containing AIV and Newcastle disease virus. J Vet Diagn Invest 2011; 23: 330– 332. Woolcock PR. Avian influenza virus isolation and propagation in chicken eggs. In E. Spackman, (Ed.). Avian Influenza Virus 1st ed. 2008 (pp.35–46). Totowa, NJ: Humana Press. Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am. J. Hygiene 1938; 27, 493-497. Iqbal M, Yaqub T, Mukhtar N, Shabbir MZ, McCauley JW. Infectivity and transmissibility of H9N2 avian influenza virus in chickens and wild terrestrial birds. Vet Res 2013; 44:100. Iqbal M, Yaqub T, Reddy K, McCauley JW. Novel Genotypes of H9N2 Influenza AViruses Isolated from Poultry in Pakistan Containing NS Genes Similar to Highly Pathogenic H7N3 and H5N1 Viruses. PLoS ONE 2009;4: e5788. Susta L, Miller PJ, Afonso CL, Brown CC. Clinicopathological characterization in poultry of three strains of Newcastle disease virus isolated from recent outbreaks. Vet Pathol 2011; 48: 349–360. Pantin-Jackwood MJ, DE Swayne. Pathobiology of Asian highly pathogenic avian influenza H5N1 virus infections in ducks. Avian Dis 2007; 51: 250–259. Pedersen J. Programming and result interpretation for avian influenza and avian paramyxovirus-1 Real-Time RT-PCR Protocols with applied biosystems 7500 fast real-time instrumentation. In SOP-AV-0003. Ames, IA: National Veterinary Services Laboratory, 2009. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, Lohman K, Daum LT, Suarez DL. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol 2002 ; 40: 3256–3260. Wise MG, Suarez DL, Seal BS, Pedersen JC, Senne DA, King DJ, Kapczynski DR, Spackman E. Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. J Clin Microbiol 2004; 42: 329–338. Pawar SD, Kale SD, Rawankar AS, Koratkar SS,Raut CG, Pande SA, Mullick J, Mishra AC. Avian influenza surveillance reveals presence of low pathogenic avian influenza viruses in poultry during 2009–2011 in the West Bengal State, India. Virol J 2012; 9: 151. Roussan DA, Haddad R, Khawaldeh G. Molecular survey of avian respiratory pathogens in commercial broiler chicken flocks with respiratory diseases in Jordan. Poult Sci 2008; 87: 444–448. 24. Ge S, Zheng D, Zhao Y, Liu H, Liu W, Sun Q, Li J, Yu S, Zuo Y, Han X, Li L, Lv Y, WangY, Liu X, Wang Z. Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcriptionpolymerase chain reaction in chicken eggs. Virol J 2012; 9: 128. 25. Carr JH. Inoculation time differentials for expression of interference of Newcastle disease virus by swine influenza virus in chick embryos. Trans Kans Acad Sci 1960; 63: 141– 146. 26. Pillai SP,Pantin-Jackwood M, Yassine HM, Saif YM, Lee CW. The high susceptibility of turkeys to influenza viruses of different origins implies their importance as potential intermediate hosts. Avian Dis 2010; 54: 522–526. 27. Spackman E,Gelb J Jr., Preskenis LA, Ladman BS, Pope CR, Pantin-Jackwood MJ, McKinley ET. The pathogenesis of low pathogenicity H7 avian influenza viruses in chickens, ducks and turkeys. Virol J 2010; 7: 331. 28. Swayne DE, Slemons RD. Using mean infectious dose of wild duck- and poultry-origin high- and low-pathogenicity avian infleunza viruses as one measure of infectivity and adaptation to poultry. Avian Dis 2008; 52: 455–460. 29. Kimura Y, Norrby E, Nagata I, Ito Y, Shimokata K. Homologous interference induced by a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture. J Gen Virol 1976; 33: 333–343. 30. Rott R. Molecular basis of infectivity and pathogenicity of myxovirus. Brief review. Arch Virol, 1979; 59: 285–298. 31. Suzuki Y. Receptor and molecular mechanism of the host range variation of influenza viruses. Uirusu 2001; 51: 193– 200. 32. Dortmans JC, Koch G, Rottier PJ, Peeters BP. Virulence of Newcastle disease virus: what is known so far? Vet Res 2011; 42: 122. 33. Kreijtz JH,Fouchier RA, Rimmelzwaan GF. Immune responses to influenza virus infection. Virus Res 2011; 162: 19–30. 34. Garcia-Sastre A. Induction and evasion of type I interferon responses by influenza viruses. Virus Res 2011; 162: 12–18. 35. Anestad G, Nordbo SA. Virus interference. Did rhinoviruses activity hamper the progress of the 2009 influenza a (H1N1) pandemic in Norway? Med Hypotheses 2011; 77: 1132–1134. 36. Linde A, Rotzen-Ostlund M, Zweygberg-Wirgart B, Rubinova S, Brytting M. Does viral interference affect spread of influenza? Euro Surveill 2009; 14: 19354. 37. França M, Howerth E, Carter DL, Byas A, Poulson R, Afonso CL, Stallknecht DE. Co-infection of mallards with low virulence Newcastle disease virus and low pathogenic avian influenza virus. Avian Pathol 2014; 43(1): 96-104. 38. Hanson LE, White FH, Alberts JO. Interference between Newcastle disease and infectious bronchitis viruses. Am J Vet Res 1956; 17: 294–298. 39. Gelb J Jr., Ladman BS, Licata MJ, Shapiro MH, Campion LR. Evaluating viral interference between infectious bronchitis virus and Newcastle disease virus vaccine strains using quantitative reverse transcription-polymerase chain reaction. Avian Dis 2007; 51: 924–934. 40. Turpin EA, Perkins LE, Swayne DE. Experimental infection of turkeys with avian pneumovirus and either Newcastle disease virus or Escherichia coli. Avian Dis 2002; 46: 412– 422. 41. Fawad A. Three Significant Events in the Poultry Industry, During Last Three Decades. Vetenaria 2014; 2: 6-10 42. Umair H, Waseem A, Anjum M S. Effect of Salmonella on hatchability and fertility in laying hen, an assessment. Vetenaria 2014; 2: 20-23 5