Methods to prevent PCR amplification of DNA from non-viable virus were not successful for infectious laryngotracheitis virus

Abstract

Molecular-based testing of poultry dust has been used as a fast, sensitive and specific way to monitor viruses in chicken flocks but it provides no information on viral viability. Differentiation of viable and nonviable virus would expand the usefulness of PCR-based detection. This study tested three treatments (1. DNAse, 2. propidium monoazide [PMA], 3. immunomagnetic separation [IMS]) applied to dust or virus stock prior to nucleic acid extraction for their ability to exclude nonviable virus from PCR amplification. Infectious laryngotracheitis virus (ILTV) was used as a model. These treatments assume loss of viral viability due to damage to the capsid or to denaturation of epitope proteins. DNAse and PMA assess the integrity of the capsid to penetration by enzyme or intercalating dye, while IMS assesses the integrity of epitope proteins. Treatments were evaluated for their ability to reduce PCR signal, measured as ILTV log 10 genomic copies (ILTV GC), of heat and chemically inactivated ILTV in poultry dust and virus stock. Compared to untreated dust samples, there was an overall reduction of 1.7 ILTV GC after IMS treatment (p<0.01), and a reduction of 2.0 ILTV GC after PMA treatment (p<0.0001). DNAse treatment did not reduce ILTV GC in dust (p = 0.68). Compared to untreated virus stocks, there was an overall reduction of 0.5 ILTV GC after DNAse treatment (p = 0.04), a reduction of 1.8 ILTV GC after IMS treatment (p<0.001) and a reduction of 1.4 ILTV GC after PMA treatment (p<0.0001). None of the treatments completely suppressed the detection of inactivated ILTV GC. In conclusion, treatments that use capsid integrity or protein epitope denaturation as markers to assess ILTV infectivity are unsuitable to accurately estimate proportions of viable virus in poultry dust and virus stocks.

Transmission of infectious laryngotracheitis virus vaccine and field strains: the role of degree of contact and transmission by whole blood, plasma and poultry dust

“…However, this was not supported by the findings in which infection was unsuccessful with dust or dust extracts collected from infected and clinically sick birds at 3, 7 and 14 dpe and given to 120 susceptible chicks in 6 isolators. This is in agreement with previous report by Bindari et al [ 36 ] that failed to infect cell cultures or chick embryos with qPCR positive dust extracts. These authors also found that spiking dust samples with cultured ILTV reduced the infectivity of the ILTV to chick embryos.…”

“…This is reinforced by experimental demonstration of airborne transmission of field and vaccine ILTV strains, potentially due to infective dust particles in the air [ 24 ]. In contrast, Bindari et al [ 36 ] were unable to isolate ILTV from ILTV PCR positive dust samples in either chick embryos or cell culture. Thus the role of poultry dust in the transmission of ILTV remains unresolved.…”

Viability RT-PCR for SARS-CoV-2: a step forward to solve the infectivity quandary

“…These included propidium monoazide and immunomagnetic separation tested on laryngotracheitis virus and ethidium monoazide on avian influenza virus. (11,12) It is worth to report that we also tested on inactivated SARS-CoV-2 suspensions a porcine gastric mucine in situ capture RT-qPCR, a method that was originally implemented in our laboratory for human enteric viruses. (20) Although proper SARS-CoV-2 infectious controls could not be included, those experiments resulted in inconclusive outcomes (data not shown).…”

“…(10) However, the application of such techniques for enveloped viruses has not fully elucidated as it has failed for avian influenza virus (IAV) and infectious laryngotracheitis virus (ILTV) while it has recently been optimized for porcine epidemic diarrhea coronavirus. (11–13) Nonetheless, the implementation of this technique has not been explored for SARS-CoV-2.…”

Characterization of poultry house dust using chemometrics and scanning electron microscopy imaging

“…In addition, dust has also been shown to be useful for the monitoring of pathogens that are primarily transmitted by feather dander such as Marek's disease virus ( Walkden-Brown et al, 2013 ). Respiratory pathogens such as infectious laryngotracheitis virus ( Ahaduzzaman et al, 2019 ), Newcastle disease virus and infectious bronchitis virus ( Tran et al, 2020 ) are detectable in dust samples using PCR although it is not clear whether this represents infective virus from the respiratory tract or inactivated viral nucleic acids present in excreta following passage through the gut ( Bindari et al, 2020 ; Yegoraw et al, 2020 ). It can be expected that pathogens originating in litter material (e.g., litter derived Aspergillus fumigatus ) will also be well represented in dust, however, it should be noted that the proportion of bedding in dust declined to low levels over time as observed in this study.…”