Reviewer #1: Borodin and colleagues present new PCR assays for the detection of typical exogenous Avian Leukosis Viruses present in their flocks of interest. The authors clearly present the need for PCR-based assays to detect particularly subgroups J and K, but limited data is presented as evidence for the improvements made by their tests. Tables contain redundant information, and graphical representation of reductions in overall infection rate would be most welcome. The authors need to ensure they are consistent and clear in the differences between exogenous and endogenous ALV, and that the distinction between particularly J and K needs to be made clear when discussing results. 
Of most particular concern is the repeat of an entire section of the introduction in the conclusion. This makes me doubt the integrity of the entire manuscript. 

Response: We thank the reviewer for the detailed comments on our manuscript. We address each point below.

R#1: Lines 60-2 – Make clearer the distinction between highly pathogenic exogenous viruses and the much less pathogenic endogenous group. 

Response: We modified the text accordingly, see line 14 and below (from here on, line numbers reference the new version of the manuscript).

R#1: Lines 74-5 – ALVE21/ev21 is not a gene, but an ALVE integration site, so please change this sentence. I think “ERV” will then be superfluous here, but it is the first time used (and therefore not defined). 

Response: We agree and have modified the text (line 29 and below).

R#1: Line 75 – the indicated reference 16 doesn’t talk about ALV susceptibility – is there an error here? Other works by Smith, Levin, Fadly etc from the early 1990s do talk about the importance of ALVE sequences (and not just ALVE21, but also ALVE6, ALVE9 etc) for receptor interference, but the relevance of receptor interference depends on the subgroup-specific viral entry receptors. 

Response: We thank the reviewer for catching this mistake. We substituted the correct reference.

R#1: Lines 112-113 – I don’t yet understand how you can eradicate an exogenous virus by PCR alone? Unless eradicate just means to identify infected individuals, rather than eradicating the impact from the flock entirely?

Response: We identified and removed infected individuals. We endeavored to make this clearer in the text.

R#1: Line 123,125 – GenBank, rather than GeneBank

Response: Thank you, fixed.

R#1: Line 133 – The ALV-J envelope is derived from an EAV-HP element, often numerous in the genome. What precautions were taken to ensure other, non-ALV chicken ERVs were not detected?

Response: The ALV J envelope is only partially derived from the EAV-HP element. The other part contains a portion of ev-D. Our primers flank the breakpoint, eliminating potential non-ALV J and ERV amplicons. We now illustrate this in the new Figure 1A.

R#1: Lines 170-2 – to put these types of figures on detection is interesting, but could it have context? Can you compare this directly to existing PCR and non-PCR approaches? 
OK, lines 177-179 do some comparison, but you say “in your hands” – can you give context of what has previously been reported in terms of sensitivity?

Response: We did not compare PCR and non-PCR methods for most of the experiment because the latter are unacceptably labor intensive for our large-scale applications (we added a statement to this effect on line 130). We synthesized the primers published by Qin et al. and repeated ALV detection according to their methods, but on our material (this is the meaning of "in our hands"). Our results are similar to those published by Qin et al. We added relevant clarification on line 132 and below. We did confirm PCR-negative birds using ELISA (line 179 and below) at the end of our eradication program.

R#1: Lines 181-185 – 52% seems very high! Is this what you expected? Or higher than you suspected?

Response: Given published estimates of ~20% overall flock mortality and the fact that not all infected birds die, our results seem in line with previous experience. We added discussion of this point on line 145. We were unable to find published PCR-based data on infection frequencies, however. 

R#1:  Also (line 183) – switch herd for flock.

Response: Done, thank you for catching this.

R#1: Line 186 – were there any differences in detection prevalence or apparent sensitivity between tissues?

Response: We did not do any among-tissue comparisons because our main goal is a non-invasive quick detection method. Since using feathers proved to be effective, we focused on them because they are the easiest to sample.

R#1: Line 204-5 – Is the 77th generation what the authors mean? 77th round of PCR testing? No data is shown to support generational crosses?

Response: Yes, we are referring to the 77th generation from the start of the breeding program. We made it clearer in the text (line 163).

R#1: Lines 235-247 (in conclusion) are a direct repeat of the introduction, only skipping the week number in each part of the third sentence. This is a very very odd thing to do – why have the authors done this? It makes me doubt other parts of the manuscript, if copy and pasting is being used

Response: We thank the reviewer for catching this unfortunate mistake. The second instance of the paragraph has been removed.

R#1: Lines 248-9 – the authors don’t appear to show at any point any difference in detection between multiple site probes? So how can this assertion be made? It could be that one probe set detects all the time?

Response: We discuss this in the paragraph starting from line 164. We conducted separate experiments, adding the JJPLN probe and the LTR locus on top of the initial JNP probe, with increased detection at each step.

R#1: Tables1-4 – lots of the primers are repeated, creating superfluous content. Better to have one large table showing how different primers are used together and for what purpose, than many separate tables with duplicated information. A single landscape orientation page would cover it. Table legends could be more informative.
Table 5 give lots of information, but it’s really had to get the message across. A graph depicting the infection rate would be much more informative, or proportional ratios as a graph.

Response: We consolidated all the primer information into one table (new Table 1) and added a figure with infection rates (Figure 2). We did, however, retain the eradication data table, in case some readers would like to see the raw numbers.

Reviewer #2: In the manuscript entitled “Complete eradication of avian leukosis virus subgroups J and K using multilocus qRTPCR in broiler cross chickens”, the authors developed a novel qRT-PCR assay to ALV subgroups A, B, J, and K . The authors worked hardly to validate their assays but the following points should be considered and addressed by the authors in the manuscript prior to being submitted for publication as detailed below:

Response: We thank the reviewer for taking the time to provide a detailed critique of our manuscript. We respond to each point below.

R#2: -The M&M is very short and the authors should expand their methods to show for example: alignment figure where they highlight the locations of primers/probe. 

Response: We provide additional details in the M&M section. We added Figure 1, the A panel shows a schematic representation of our probe position.

R#2: -In DNA isolation and qRTPCR: how many feather samples were used? How many samples were used for RNA isolation? How many field samples? Only 10?

Response: We used 16 feathers for RNA isolation (now mentioned in M&M, line 98). All samples were field samples.

R#2: -Description for how the authors set their detection limit for each subgroup assay is unclear. How genome copies equivalent was calculated?

Response: We used cloned virus fragments from the relevant subtypes. In the case of ALV J, we also used the published test system (Qin et al., 2013) to verify our estimates.

R#2: -As the virus can be detected in other sources than feathers (for example eggs), why the authors didn´t test their assays against egg sample, for example? 

Response: Our aim was to develop a non-invasive system, so we did not use eggs. We did, however, use several tissues in our regional survey (line 145 and below).

R#2: -Did the author validate their new assays on Egg samples? 

Response: See above.

R#2: -Did the author test their assays against mixed sample (different ALV subgroups in one sample)? 

Response: We used field samples, a portion of which were double infected by ALV J and K. This is now mentioned on line 140).

R#2: -Can the authors provide few PCR curves for the results of their assays? 

Response: We added a representative set of curves in the B panel of Figure 1.

R#2: -Title: "complete eradication" can be removed. It is just an opinion of this reviewer that here the authors developed a tool which can be useful for control and prevention of ALV.

Response: Since we can no longer detect the virus in our flock, we believe "eradication" is an appropriate term. However, since our system has a finite precision, we agree with the reviewer that we cannot state that the eradication is complete. We dropped that word from the title.

R#2: -Abstract and M&M: real-time quantitative reverse transcriptase PCR or quantitative real-time PCR?

Response: It is quantitative real-time PCR. To avoid confusion, we no longer use the abbreviation in the text.

R#2: -Line 31: “two genetic subgroups” please name them.

Response: Changed the text accordingly.

R#2: -Line 52: Please add reference.

Response: Added (it is reference [1]).

R#2: -Line 60: where is group F circulating? In which species?

Response: This subgroup is specific to pheasants (we added a reference to that effect on line 17).

R#2: -Line 100, 117 and the rest of the manuscript: “animals” to birds

Response: Fixed, thank you.

R#2: -Line 143: can move this paragraph after line 155.

Response: We moved the paragraph as the reviewer suggests.

R#2: -Line 145: 50 cycles? How could the authors exclude that this large number of cycles generate false positive results?

Response: We used fresh reagents and negative controls, including specific pathogen-free chicken DNA (now mentioned on line 137), to check for false positives. We also note that false positives are less detrimental than false negatives when the goal is to eliminate infected individuals.

R#2: -Line 151: the abbreviations for the primers/probe should be clarified either at the M&M or below the tables.

Response: Primer and probe names are arbitrary and are not acronyms.

R#2: -Line 179: “SPF” abbreviation was not mentioned in the manuscript.

Response: "Specific Pathogen-Free." Now written in full (line 137).

R#2: -Line 187: “We then moved on to analyze DNA from several tissues (feathers, liver, spleen, 
etc.)” what is etc? please name explicitly the tissue. 

Response: All tissues now explicitly stated.

R#2: -Line 28: qRT-PCR?

Response: We no longer use the abbreviation.

R#2: -Table 1: why ALV B is after ALV J?

Response: Primers for all subgroups are now listed alphabetically in the table.

R#2: -Table 2: as those primers/probes are already published, why the authors mentioned them in a separate table? Did the authors make any modification?

Response: We re-synthesized the primers, using the exact published sequences. We list them here (new Table 1) so that the readers do not have to search through references for them and can verify that we in fact used the correct primers.