Phylodynamics of Highly Pathogenic Avian Influenza A(H5N1) Virus Circulating in Indonesian Poultry
, Rogier Bodewes 3, Restuadi Restuadi 4, Alex Bossers 1,5, Agustiningsih Agustiningsih 6, Jan Arend Stegeman 1, Guus Koch 7 and David Handojo Muljono 8,9,10,*Round 1
Reviewer 1 Report
Overall the manuscripts provide new information but need significant revision.
There are many points that could be expanded, statements were very vague and didn't really make any ground-breaking conclusions.
The Authors don't really drill home how their data is novel - how the H5 AIVs in Indonesia are evolving through persistence, with little to no external introduction.
There is no mention of wild bird involvement, despite the huge dataset or vaccination programs.
The figures need to be edited as they are stretched and hard to read or the text is on a dark background.
A few grammar mistakes here and there.
This manuscript would benefit greatly from further analysis of the key molecular markers that are driving evolution and if there were key reassortment events, and if wild birds are driving such persistence in Indonesia. The authors mention that this could be a cause, similarly to fomites/humans but do not go into detail. It feels like it's missing that extra step. Although still provide basic data but could be better with further analysis of sequence data describing the key changes in the gene segments that potentially carry an impact on virus phenotype.
Some suggestions and typos are indicated in the attached manuscript pdf file.
Comments for author File: 
Comments.pdf
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 2 Report
The manuscript of Karo-karo et al. aims at phylogenetic analysis of HPAI H5N1 viruses detected in Indonesia in 2013-2016. The study shows evolution of Indonesian H5N1 viruses into multiple subclades. There are several issues which need to be addressed by the Authors to improve the quality of the study.
1. According to Materials and Methods the Authors performed BEAST analysis with various sequence sets, first for Indonesian viruses from 2010-16, then separately for clade 2.1.3.2, 2.1.3.2a, 2.3.2.1c from 2006-16; then for Indonesian viruses from 2003-2016; and finally for all H5 sequences available in databases. However, it is unclear to which dataset refer the presented results. For example, in Tables 1 and 2 there are “geographical zones” such as Worldwide HA (H5) and Worldwide HA (H5) + Indonesian HA 2015-2016. So, the dataset “Worldwide HA (H5)” did not contain Indonesian H5 from 2015-16? Why these years were excluded? Why were these separate analyses performed?
2. It is unclear why a separate analysis for only Indonesian H5 sequences was performed. This dataset contains gaps which leads the Authors to some peculiar conclusions that clade 2.3.2.1c evolved from the clade 2.1.1. (lines 177-178, 248-249). Both clades share a common ancestor but there are long sampling gaps and this dataset lacks intermediate sequences which is clear from the tree for the “worldwide HA”. However, in other part of the manuscripts the Authors claim that 2.3.2.1c evolved from 2.3.2.1b.
3. The bias introduced by different sequence datasets is also clear from the different topologies of the Indonesian groups seen in the tree containing only Indonesian sequences (Fig. 1) and the tree for the H5 detected in the world (Fig. 3). Especially, for the 2.1 subclades.
4. What is the basis for conclusion that clade 2.2 evolved from 2.5, 2.1 and 2.5 from 2.4, and the latter from clade 1? Do the Authors take into account the posterior probabilities when inferring conclusions?
5. According to the caption of Figure 1 and data in Tables 1 and 2, sequences from 2003-2016 were analysed but from the description in the tree it appears that the oldest sequences came from 2005. In line 201 it is mentioned “over the course of 11 years”.
Minor comments:
1. According to Table 2 the MRCA for Indonesian HA (2003-2016) existed in 2002 but in Fig.1 the MRCA locates before 2000.
2. Lines 181-184 – the description is confusing and chaotic. It would be more clear if the existence of various clades was described in chronological order.
3. Discussion – lines 263-266 – please mention the substitution rates reported in the cited studies. Study by Lewis et al. was performed for wild-bird LPAI viruses of all known subtypes so comparing substitution rates seems pointless.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
