Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption

Reverse transcription-polymerase chain reaction (RT-PCR) has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV). One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP) gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs). The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses.


Introduction
Newcastle disease is a highly contagious disease of birds affecting many domestic and wild avian species. The high susceptibility and mortality often cause epidemics in poultry populations. Transmission occurs by exposure to fecal and other excretions from infected birds, or through contact with contaminated feed, water, equipment and plumage [1]. Hemagglutionation (HA) and ELISA methods have been applied to detect NDVs in chicken embryos and tissues [2]. Although the egg passage test is more sensitive than HA and ELISA [3], it will take several days to obtain results, therefore, a rapid and sensitive method will be helpful to detect virus in varied samples.
RT-PCR has been developed to detect and pathotype Newcastle disease virus (NDV) in clinical samples [4][5][6]. However, RT-PCR has limited sensitivity in detecting complicated samples, such as feces, tissue samples and contaminated water. Thus, an effective and simple virus concentration and purification method will be great helpful to enhance the detection sensitivity [7,8].
Newcastle disease virus, being a paramyxovirus, causes hemagglutination of chicken red blood cells. There are two viral glycoproteins, HN and F fusion protein, embedded in the viral envelope, HN is responsible for virus attachment to host cell receptors, and the F fusion protein mediates fusion of viral envelope with the host cell membrane, enabling the virus to enter host cells [9]. By virtue of the property of NDV binding to red blood cells (RBCs), we concentrated and purified NDV by a red blood cell adsorption and de-adsorption process, we optimized the conditions of adsorption and de-adsorption to develop an effective method for NDV purification and detection. In this study, one pair of primers was designed according to the relevant sequence of the nucleocapsid protein (NP) gene of NDV acquired from Genbank (accession number FJ766531). We applied the reverse transcription (RT)-polymerase chain reaction (PCR), coupled with RBC adsorption to detect NDV in varied samples. The results of this study demonstrated that the described RBC adsorption RT-PCR assay has the potential to be used for the rapid and sensitive detection of NDV isolates in a variety of samples, and could be used to detect other viruses with the property of hemagglutination.

Virus strains and virus propagation
The La Sota strain came from the commercial vaccine strain. The F48E9 strain was obtained from China Institute of Veterinary Drug Control. The N79 strain was preserved by our laboratory. All the strains were amplified in ten-day-old embryonated SPF eggs

Preparation of red blood cells
Venous blood (0.5 ml) was collected from 2 month-old healthy chickens with sodium citrate anticoagulant, centrifuged at 750× g for 5 min, the serum and buffy coat were aspirated, the remaining erythrocytes were washed with phosphate buffered saline (PBS) three times by spinning at 750× g for 5 min, then the cells were suspended in 200 μl PBS.

Efficiency of red blood cells Adsorption
The allantoic fluid (1.0 ml 5 × 10 9 EID 50 ) was mixed with 50 μl washed red blood cells, and incubated with rotation (150 rpm) at room temperature for 20 min, After adsorption treatment, The cells were then collected by spinning at 750× g for 5 min. The virus titer in supernatant was tested by the EID assay.

Optimization of the conditions releasing NDVs from RBC
After NDVs adsorption, the collected cells were suspended with 1 ml saline containing different concentrations of EDTA and β-mercaptoethnol for release of NDV from RBC at 37°C, then spin at 1000χg for 5 min. The supernatant was collected and tested by the EID assay.

Determination of sensitivity of the RBC coupled RT-PCR
To estimate the sensitivity of the RBC coupled RT-PCR in comparison with regular RT-PCR, Serial 10-fold dilutions of the 1 × 10 9 EID 50 NDVs allantoic fluid were made in PBS buffer. RNA was extracted from 1 ml aliquots of each dilution by red blood cell adsorption approach and regular approach, respectively. The RNA extracts were simultaneously analyzed in parallel by RT-PCR.

Detection of NDV in fecal samples
The feces (0.2 g) collected from F48E9 infected chickens, and diluted the feces by 1000 and 10000 fold, then centrifuged at 15,000× g for 10 min two times, the supernatant was incubated with 50 μl washed RBC to adsorb NDV particles with rotation (150 rpm) at room temperature for 30 min, the cells were collected by spinning at 750× g for 5 min. After de-adsorption of the NDVs from cells, the supernatant was tested for the presence of NDV with RT-PCR.

Detection of NDV in different tissue samples by the RBC coupled RT-PCR
To verify whether the approach was applicable to detect complicated samples, liver, kidney, and lung samples were collected from N79 infected chickens and homogenized in 1 ml PBS, then centrifuged at 15,000× g for 10 min two times. After adsorption and de-adsorption process, the samples were tested in parallel by RT-PCR.
Detection of NDV in infected chickens with different NDV strains 60 4-week-old SPF White Leghorn chickens were hatched and reared in isolation, 15 chickens per group were inoculated with 2 × 10 6 EID50 of the following strains of NDV: La Sota, F48E9 and N79, and kept in isolation, four days post inoculation, feces were collected, spleen, brain, and kidneys were also aseptically collected. All the samples stored separately at 70°C until further analysis. Homogenized fecal and tissue samples (0.2 g) from infected chickens were suspended in 1 ml PBS, then centrifuged at 15,000× g for 10 min two times, After adsorption and de-adsorption of the NDVs from red blood cells, the supernatant samples were tested in parallel for the presence of NDV with RT-PCR.

Efficiency of red blood cells adsorption and de-adsorption
After treating 1.0 ml allantoic fluid (5 × 10 9 EID 50 ) with 50 μl washed red blood cells, almost no viruses could be detected in the supernatant by HA experiment, most of NDVs attached to the surface of red blood cells. After NDVs adsorption, we optimized the de-adsorption conditions by suspending the collected cells in saline with different concentrations of EDTA and β-mercaptoethnol, the results showed 5 mM EDTA and 10 mM β-mercaptoethnol could effectively release NDVs from red blood cell at 37°C, while high concentrations of EDTA and β-mercaptoethnol will lead to hemolysis and low virus titer in the supernatant ( Table 1).

Sensitivity of the red blood cell coupled RT-PCR
To assess the sensitivity of red blood cell adsorptioncoupled RT-PCR, we serially diluted the NDV allantoic fluid. The limit of NDV detection was in the range of 10 7 fold diluted samples (100 EID 50 ) (Figure 1), which is much more sensitive than the regular RT-PCR on agarose electrophoresis detection.

Sensitivity of NDV detection in fecal samples
The efficiency of the established RT-PCR assay was assessed in diluted fecal samples from infected chickens. After gel electrophoresis, a 578 bp band was visible until the 10 4 fold dilution (Figure 2), therefore, RT-PCR, coupled with red blood cell adsorption, is much more sensitive than the regular RT-PCR in detecting NDVs feces.

Detection of NDV in different tissue samples
To assess whether the approach is applicable to tissue samples, the liver, kidney, and lung samples from chickens infected with N79 NDVs were screened by RT-PCR after the adsorption and de-adsorption process ( Figure 3). The positive RT-PCR in each samples demonstrated that the approach was applicable to detect NDVs in different kind of samples.
The results showed the new approach could detect different NDV strain in all the varied samples, while regular RT-PCR only detected part of the samples. This demonstrates that red blood cell coupled RT-PCR is a rapid and efficient method for the detection of NDV in varied strains as well as varied samples.

Discussion
NDV has a variety of strains that differ widely in virulence, from causing an asymptomatic infection to lethal disease. Although vaccination programs have provided significant protection against NDV outbreaks, infections by ND viruses have been reported frequently around the world in recent years. Correct and fast diagnosis of NDV will surely help in effective controlling the disease. Unfortunately, rapid detection of NDV at the onset of the disease has always been hampered by the lack of a sensitive and fast detection method. Current diagnosis of NDV infection by conventional virus isolation and serological tests, such as haemagglutination inhibition (HI) and serum neutralization, are either time-consuming or lack the required sensitivity [10][11][12].
Feces from NDV infected birds play an important role within the spread of NDV, however, the bacterial contamination and toxic substances in feces limit NDV detection by virus isolation in embryonated eggs, therefore, RT-PCR is a valuable alternative. It has been reported that some organic substances present in feces and tissue samples would inhibit the activity of reverse polymerase and Taq polymerase [13]. Furthermore, the large amount of celluar RNA in tissue samples will interfere with the RT-PCR reaction, usually leading to false positive and negative results, so the property of the samples is critical for obtaining RNA of good quality, however, the conventional RNA extraction methods couldn't get rid of these problems, rather sometimes facilitate contamination and loss of virus particles during sample preparation, especially due with low viral load samples [14].
In this study, we concentrated and purified NDVs by a red blood cell adsorption and release process depending on the ability of NDV binding to RBC, we could thus get rid of the contaminant substances and cellular RNA. This approach has been proved to be much more sensitive than the regular RT-PCR in treating varied samples and different NDV strains. Therefore, combining the RT-PCR and red blood cell adsorption is useful when the amount of NDV particles is too low to be detected during the onset of an outbreak, or possibly when it is necessary to trace the origin of the different viruses. In recent years, duplex polymerase chain reaction, real time PCR, and molecular probes have been developed to detect NDVs in different samples with high sensitivity [15,16], when combination of these new PCR methods with the red blood cell purification procedure will greatly enhance the detection sensitivity and decrease false results.